Use of the antibiotic, olivomycin, for the cytochemical study of chromatin]

The possibility to use the antibiotic olivomycin for fluorescence cytochemistry of chromatin properties has been shown. It is found that the binding of olivomycin to nuclei reveals the functional state of chromatin and changes in the course of its activation. The essential condition for the application of the method described is the use of ethanol-aceton fixative. When other fixatives are used, in particular 70% ethanol, olivomycin binding reflects differences in nuclear DNA amount, rather than those in chromatin properties. The advantages of the method described, in comparison with the commonly used technique, are associated with the high affinity of olivomycin to DNA, absence of olivomycin binding with RNA, simplicity of the staining procedures , and with rather a high stability of complexes formed between olivomycin and DNA.     

Effect of the diuretic diacarb on the antitumor activity of the antibiotics rubomycin and olivomycin]

Combined use of rubomycin and olivomycin with diacarb in treatment of rats with Pliss lymphosarcoma increased the antiblastomic activity of the antibiotics. The antitumor effect of rubomycin and olivomycin was increased by diacarb in the same degree as that of dipin, a synthetic cytostatic.     

Myeloinhibitory effect of the action of some antitumor antibiotics and its comparative assessment]

General toxic and myeloinhibitory effects of some antitumor antibiotics, such as rubomycin, olivomycin, bruneomycin and karminomycin administered intraperitoneally in a single LD50 to mice were studied. It was found that the general toxicity of bruneomycin and karminomycin was almost the same and 5 to 8 times higher than that of rubomycin and olivomycin. The use of the above antibiotics resulted in definite shifts in the blood systems of healthy mice. The most significant suppression of hemopoesis accompanied by a pronounced depression of the number of the myelocariocytes was observed after the use of olivomycin. The effect of karminomycin was characterized by suppression of erythro-, myelo- and lymphopoesis and depression of the number of the granulocytes and lymphocytes of the blood. Bruneomycin and rubomycin had a short-time myeloinhibitory effect. The erythroid cords of the bone marrow proved to be most sensitive to the inhibitory effect of the antibiotics. However, inhibition of the erythropoesis accompanied by deep reticulocytopenia did not induce the respective depression of the erythrocyte number. The lymphoid cords was in the 2nd place by its sensitivity to the antibiotics and the myeloid and megocariocytal cords were in the 3rd and the 4th places respectively. Complete reduction of hemopoesis in the animals was observed by the 10th day of the drugs use.     

Modification of olivomycin A at the side chain of the aglycon yields the derivative with perspective antitumor characteristics

A novel way of chemical modification of the antibiotic olivomycin A (1) at the side chain of the aglycon moiety was developed. Interaction of olivomycin A with the sodium periodate produced the key acid derivative olivomycin SA (2) in 86% yield. This acid was used in the reactions with different amines in the presence of benzotriazol-1-yl-oxy-trispyrrolidino-phosphonium hexafluorophosphate (PyBOP) or diphenylphosphoryl azide (DPPA) to give corresponding amides. Whereas olivomycin SA was two orders of magnitude less cytotoxic than the parent antibiotic, the amides of 2 demonstrated a higher cytotoxicity. In particular, N,N-dimethylaminoethylamide of olivomycin SA showed a pronounced antitumor effect against transplanted experimental lymphoma and melanoma and a remarkably high binding constant to double stranded DNA. The therapeutic effects of this derivative were achievable at tolerable concentrations, suggesting that modifications of the aglycon’s side chain, namely, its shortening to methoxyacetic residue and blocking of free carboxyl group, are straightforward for the design of therapeutically applicable derivatives of olivomycin A.     

Pathomorphological picture of the testes of mice administered antitumor antibiotics and their comparative evaluation

A single administration of the LD50 of bruneomycin, carminomycin, rubomycin or olivomycin and the use of the antibiotics for 10 days in a dose 2 times higher than the therapeutic one and amounting to 10 per cent of the LD50 induced dystrophic and destructive changes in the sexual glands of mice. The changes in the testis were more pronounced after the single use of the antibiotics. Recovery processes were observed in the testis beginning from the middle of the third week after the use of carminomycin and rubomycin and from the end of the second week after the use of olivomycin. Bruneomycin was an exception: the pronounced destructive changes in the sexual glands after its single or repeated use persisted within the observation period.     

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A-T and G-C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another. —Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G-C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A-T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A-T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G-C specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.—The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.     

Chromomycin, mithramycin, and olivomycin binding sites on heterogeneous deoxyribonucleic acid. Footprinting with (methidiumpropyl-EDTA)iron(II)

The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA)iron(II) [MPE x Fe(II)] DNA cleavage inhibition pattern technique. Two DNA restriction fragments 117 and 168 base pairs in length containing the lactose operon promoter-operator region were prepared with complementary strands labeled with 32P at the 3' end. MPE x Fe(II) was allowed to partially cleave the restriction fragment preequilibrated with either chromomycin, mithramycin, or olivomycin in the presence of Mg2+. The preferred binding sites for chromomycin, mithramycin, and olivomycin in the presence of Mg2+ appear to be a minimum of 3 base pairs in size containing at least 2 contiguous dG x dC base pairs. Many binding sites are similar for the three antibiotics; chromomycin and olivomycin binding sites are nearly identical. The number of sites protected from MPE x Fe(II) cleavage increases as the concentration of drug is raised. For chromomycin/Mg2+, the preferred sites on the 70 base pairs of DNA examined are (in decreasing affinity) 3'-GGG, CGA greater than CCG, GCC greater than CGA, CCT greater than CTG-5'. The sequence 3'-CGA-5' has different affinities, indicating the importance of either flanking sequences or a nearly bound drug.     

Parameters of the toxic action of antibiotic derivatives of aureolic acid in acute and subchronic experiments]

The parameters of the lethal effect of aureolic acid derivatives, such as mithramycin, variamycin and olivomycin were studied on mice, rats and rabbits. As for the most of the administration routes the first two drugs were characterized by irregular distribution of resistance to thier lethal effect, which was mathematically expressed by polymodality of the dose-response curve. The above drugs were characterized by cumulative properties. The toxicity parameters depended on the animal species and administration route.     

Pathomorphological picture of the liver in mice administered antitumor antibiotics intraperitoneally and its comparative assessment]

A single administration of carminomycin, ribomycin or olivomycin in LD50 or treatment of the experimental animals with these antibiotics for 10 days in the therapeutic doses equal to 10 per cent of the LD50 induced distrophic and necrobiotic changes in the liver. The use of bruneomycin in the equivalent doses induced sclerotic process in addition to the above doses resulted in a decrease in the colour intensity of DNA, RNA and protein as compared to the control, the content of glycogen and a marked increase in the amount of lipids in the hepatocyte cytoplasm. The most pronounced shifts were observed with the use of carminomycin, rubomycin and especially bruneomycin in single doses. With the use of olivomycin in a single dose the shifts were less pronounced. It should be noted that with the use of carminomycin and rubomycin the damages were of the same character by their intensity. The changes in the liver on the use of carminomycin, rubomycin and olivomycin in single doses or during the treatment course were reversible, while on the use of bruneomycin they preserved to the end of the experiment.     

Studies with a fluorescent vital probe for DNA in mammalian cells

Olivomycin is taken up efficiently by HeLa cells and by rat fibroblast cells at 38.5 °C, but not by BHK cells. On irradiation with light of 425 nm wavelength, the nuclei of living cells that have taken up olivomycin fluoresce. When olivomycin complexes with DNA in solution, the emission spectrum broadens and shifts, the excitation wavelength maximum shifts up 15 nm, and the fluorescence polarization increases. In HeLa and fibroblast cells, the fluorescence characteristics indicate that olivomycin is entirely complexed to DNA, and its rotational mobility indicates that it is complexed to DNA in regions where other components of the chromatin offer no steric hindrance.     

Results of determining the sensitivity of hypernephroid cancer to cytostatic agents in vitro and clinically]

Sensitivity of the cells of 57 human hypernephromas was determined with respect to 8 drugs, i.e. tiodipin, fluorbenzotef, mithramycin, cyclophosphan, tiotef, rubomycin, 5-fluoruracyl and olivomycin with the help of the method of primary plasma cultures. Because of the changes introduced earlier into the estimative concentrations used in vitro, coincidence of the results of the sensitivity determined in vitro with the results obtained during the treatment of the same patients in clinics, significantly increased (up to 90 per cent).     

Effect of olivomycin on denervation changes in the membrane of frog tonic muscle fibers]

Membrane properties of frog denervated tonic muscle fibers were investigated after the action of olivomycin, which is an inhibitor of protein synthesis. Olivomycin injected as a single dose (7.5 mg) during 1--2 days after the denervation of m. pyriformis decreases the regenerative action potentials. This action is decreased three weeks after denervation. After denervation, olivomycin failed to prevent the appearance of extrajunctional cholinoergic receptors, but blocked the increase of junctional ones. The data support the view that some neuronal factor may influence the synthesis of functional membrane proteins.     

Inhibition of the c-Myc Oncogene by the Aureolic Acid Group Antibiotics

Doklady Biochemistry and Biophysics - GC-rich stretches in the DNA minor groove are the established intracellular targets for the aureolic acid group of antibiotics such as olivomycin A and its...     

Use of the DNA-specific fluorochrome olivomycin for work with cell cultures]

A high-sensitive, easy and rapid proximate method has been developed to reveal mycoplasmas in the monolayer cell cultures using home fluorescent antibiotic olivomycin. This method has been used to screen many cell lines with its high effectiveness being shown. As based on this method the following control methods are worked out: indication of mycoplasmas in animal blood sera, used for the cultivation of cell lines detection of contaminant microorganisms of the cell cultures (fungi and bacteria) and human ureaplasmas.     

Species specific differences in the toxicity of mithramycin, chromomycin A3, and olivomycin towards cultured mammalian cells

Three structurally related anticancer drugs, mithramycin, chromomycin A3, and olivomycin, showed large unexpected differences (up to more than 1000 fold) in their toxicity towards cultured cells from various species (human, Chinese hamster, Syrian hamster, and mouse). Among the cell types examined, human cells (both a diploid fibroblast cell strain and HeLa cells) were maximally sensitive to all these drugs, followed by the Syrian hamster kidney cells (BHK 21). The mouse (LMTK- cells) and Chinese hamster (CHO) cells, which were more resistant, showed interesting differences in their sensitivity towards these drugs. For example, whereas the mouse cells were more resistant to mithramycin than CHO cells, the sensitivity pattern was reversed for both chromomycin A3 and olivomycin. In cell extracts derived from human, mouse, and Chinese hamster cells RNA synthesis, which is the cellular target of these drugs, showed identical sensitivity to both mithramycin and chromomycin A3, indicating that the species specific differences in the toxicity to these drugs are at the level of cellular entry of these compounds. Based on the structures of these glycosidic antibiotics and their patterns of toxicity, it is suggested that the intracellular transport of these drugs involves specific interactions between the sugar residues on these compounds and some type of cell surface receptor(s), which differ among different cell types. Some implications of these results for toxicity studies are discussed.     

Action of aureolic acid and dactinomycin group antibiotics on human brain tumors in culture]

The cytotoxic effect of antitumour antibiotics, such as dactinomycin, mithramycin, variamycin and olivomycin on the cells of the human brain tumours (multiform glyoblastoma, arachnoidendotelioma and astrocytoma) grown by the method of the primary plasmic culture was studied. Dactinomycin was superior to the antibiotics of the aureolic acid group in the rate and level of the cytotoxic effect on the tumour cells: 76 per cent of the above tumours were sensitive to dactinomycin, 56 per cent to mithramycin and 52 per cent to variamycin and olivomycin. Among the total number of the tumours sensitive to the drugs the number of the highly sensitive tumours amounted to 57.9 per cent for dactinomycin and 30.8--38.5 per cent for the antibiotics of the aureolic acid group. Definite differences in the efficiency of the antibiotics of the aureolic acid group with respect to different types of the brain tumours were observed.     

Characteristics of interstitial cells of the adrenal medulla after stimulation of prostaglandin production by lasix

The paper presents structural characteristics of interstitial cells after a single or multiple administration of lasix. The single dose stimulated the process of prostaglandins synthesis in the interstitial cells of Kidney papillae. The prolonged administration of lasix led to relative decrease in prostaglandin production if compared with that of a single dose. Besides, there was noted formation of these cells second population, which elaborated the connective tissue proteins and glycosaminoglycans. It was suggested that the prolonged administration of lasix effected the interstitial cells causing inanition of their prostaglandin synthesizing ability and stimulating processes of production of the connective tissue intercellular substances by them.     

Pharmacokinetics of C14-olivomycin in the body of mice with lymphosarcoma (L10-1 strain)]

The pharmacokinetics of C14-olivomycin after its single intravenous administration to mice with lymphosarcome (LIO-I) was studied. It was shown that according to the specific radioactivity the organs may be placed in the following order: I hour after the antibiotic administration-the blood, liver, spleen, thymus, tumor, muscle; 3 hour after the administration-the liver, spleen, thymus, blood, tumor, muscle. Accumulation of olivomycin in the mouse organs was mainly in direct dependence on the dose of the antibiotic administered. Chromatography of the substances extraceted with ethylacetate from the urine collected at various periods after C14-olivomycin administration showed the presence of a new radioactive product (Rf 0.35-0.37) in addition to the unchanged antibiotic (Rf 0.53). Bioautographic analysis of the chromatograms showed that the product of C14-olivomycin conversion preserved its biological activity. The analysis of the substances extracted with ethylacetate from the liver, spleen and tumors 3 hours after the antibiotic administration reveiled (except of the liver) the presence of a spot with Rf corresponding to that of the initial drug.     

Role of antibiotics in the ecology of actinomycetes]

The organisms producing ristomycin, rifamycin, lincomycin, tobramycin, rubomycin carminomycin, olivomycin, bleomycin and actinomycin D were practically not sensitive to their own antibiotics in the concentrations close to those produced during the biosynthetic process. At the same time other actinomycetous species close to the above organisms were suppressed to a significant extent by their antibiotics in the same concentrations. The experimental data indicated that the antibiotics had a protective effect on the organisms producing them and played a significant role in ecology of the actinomycetes.     

Stereochemistry and kinetics of interaction with DNA of the antineoplastic antibiotic olivomycin

The kinetics of interaction of antitumor glycoside antibiotic olivomycin with DNA has been investigated. The existence of two relaxation times in the experimental kinetics curves indicates that two types of antibiotic--DNA complex are formed. We have measured the rate constants of association and dissociation processes and determined their temperature dependences. It is suggested, that one of the complex form results from nonspecific interaction between glycoside residues of the antibiotic molecule and sugar-phosphate backbone of DNA whereas the other type of complex exhibits a pronounced specificity for GC-rich regions on DNA. The binding specificity probably results from formation of a H-bond between the antibiotic chromophore ring and guanine 2-amino group. A stereochemical model for olivomycin-DNA complex is proposed. According to this model the antibiotic chromophore and glycoside residues are located in the narrow groove of DNA.