Induction of action potentials in fish red muscle by denervation

The effects of denervation on the electrical membrane properties of fish red muscle were investigated. Forty to fifty hours after denervation, miniature endplate potentials disappeared abruptly and field stimulation of the nerve within the muscle failed to evoke endplate potentials, indicating that transmission failure occurred at this time. The membrane resistance of the red muscle fibre increased after denervation. Normally innervated fish red muscles do not generate action potentials in response to either nerve or direct muscle stimulation. However, approximately 3 weeks after nerve sectioning, action potentials could be induced in the muscles. The action potential was sodium-dependent, and was sensitive to tetrodotoxin. Actinomycin D injected in the early phase after operation suppressed the induction of the action potential. These results indicate that RNA synthesis is preliminary to the induction of the action potential mechanism, and that this mechanism is under neural control.     

Insulin action in denervated skeletal muscle. Evidence that the reduced stimulation of glycogen synthesis does not involve decreased insulin binding

The motor nerves to rat soleus and epitrochlearis muscles were sectioned 1-3 days before the muscles were incubated in vitro to assess insulin action and binding. The hormonal stimulation of [U-14C]glucose into glycogen in both muscles was decreased by over 80% after 3 days of denervation. Associated with the reductions in glycogen synthesis were losses in the ability of insulin to activate glycogen synthase. Even so, denervated and control muscles bound the same amounts of 125I-labeled insulin over a wide range of insulin concentrations. Scatchard analysis indicates that denervation changed neither receptor numbers nor affinities. The results presented strongly suggest that the loss of the ability of insulin to stimulate glycogen synthesis following denervation is the result of a postreceptor defect.     

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 diuretics (diacarb and lasix) on the experimental antitumor activity, toxicity and pharmacokinetics of the antibiotic, olivomycin

Diacarb or lasix in combination with olivomycin increased the antibiotic antiblastomic activity and toxicity when used for the course treatment of mice with transplanted lymphosarcoma LIO-I. The chemotherapeutic indices of such combinations did not differ from those of olivomycin used alone. Diacarb decreased excretion of olivomycin with the urine in healthy mice, while lasix increased it.     

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.     

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.     

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.     

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.     

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.     

Neural control of gene expression in the skeletal muscle fibre: the nature of the lesion in the muscular protein-synthesizing machinery following denervation

Experiments are reported showing that following 8 days of denervation the function of the protein-synthesizing machinery, operating in the rat gastrocnemius fibres, is altered, probably as a consequence of decreased amounts of ribosomes and actively translated mRNA. In addition, the data obtained show that the amount per muscle and the availability per ribosome of the soluble factors involved in the process of protein synthesis are markedly decreased, thus suggesting that the amounts of ribosomes, mRNA and soluble factors are regulated in a concerted fashion when muscular protein synthesis is decreased after denervation.     

Axonal transport blockade and denervation have qualitatively different effects upon skeletal muscle metabolism

The activity and isoenzyme pattern of muscle lactic dehydrogenase (LDH) was measured at different times after axonal transport blockade by colchicine or after denervation. After denervation, total LDH activity decreased and the isoenzyme pattern was altered, LDH-1 being the most affected form. In contrast, after axonal transport blockade there was a decrease in LDH activity but the isoenzyme pattern was not modified. Denervation abolishes both nerve-evoked muscle activity and the release of neuro trophic substances from the nerve whereas colchicine blocks axonal transport without affecting the nerve capacity to conduct action potentials or neuromuscular transmission. It is then concluded that nerve-evoked muscle activity is the most important factor in the regulation of muscle LDH isoenzyme distribution. On the other hand, muscle metabolism can also be regulated by axonally transported molecules. The results presented here show that there is a qualitative difference between the effects of denervation and those of axonal transport blockade upon the muscle, since only denervation altered the isoenzyme pattern of muscle LDH.     

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.     

Action of hepatic chalones on the proliferation of hepatocytes from intact and denervated livers

The paper is concerned with the action of chalones, tissue-specific inhibitors of cell proliferation, on DNA synthesis and mitotic activity of hepatocytes in the intact and denervated liver during regeneration. Experiments were made on Wistar rats. Liver denervation was performed by bilateral subdiaphragmal vagotomy. In control and vagotomized animals, two thirds of the liver was resected. The data obtained indicate that chalones noticeably reduce the number of DNA-synthesizing cells and mitoses in the regenerating liver of intact animals. During regeneration of the denervated liver, chalones do not produce any inhibitory action on the intensity of proliferation. Analysis of the data obtained allows a conclusion that preservation of adequate innervation of the organ is needed for realization of the action of hepatic chalones.     

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.     

Increased Cytosolic Androgen Receptor Binding in Rat Striated Muscle Following Denervation and Disuse

Abstract: We investigated the effects of denervation and disuse on cytosolic androgen receptor binding by rat striated muscle. Denervation of the extensor digitorum longus and tibialis anterior muscles caused a 40–50% increase in cytosolic androgen receptor concentration with no change in apparent binding affinity. This effect was evident at 6 h postdenervation, maximal at 24 h, and declined to 120% of the control level 72 h after denervation. A 40% increase in cytosolic androgen receptor concentration was also noted 24 hr after denervation of the hormone-sensitive levator ani muscle. The effect of denervation on androgen receptors was not blocked by in vivo injection of cycloheximide; therefore, de novo receptor synthesis probably is not involved in the observed increase. Disuse, produced by subperineurial injection of tetrodotoxin into the tibial and common peroneal branches of the sciatic nerve, mimicked the effect of denervation on androgen receptor binding, suggesting that neuromuscular activity is important in regulation of receptor concentration. Possible mechanisms subserving this effect are discussed.     

Studies on the regulation of beta-nerve growth factor gene expression in the rat iris: the level of mRNA-encoding nerve growth factor is increased in irises placed in explant cultures in vitro, but not in irises deprived of sensory or sympathetic innervation in vivo

Beta-nerve growth factor (NGF) is a protein necessary for the survival and maintenance of sympathetic and sensory neurons that appears to be produced by the target tissues of these neurons in vivo. Both denervation and the culture of explants of one model target, the rat iris, leads to an increase in the NGF content, suggesting that innervating neurons may regulate a step in synthesis or turnover of NGF. To determine whether there is a change in synthesis controlled at the mRNA level, the rat iris has been assayed for its content of NGF mRNA after surgical and chemical denervation and after explant into culture. Using a sensitive blot hybridization assay, a large, rapid increase in the content of NGF mRNA was observed upon explant of the rat iris. The increase was readily detectable within 1 h, reached a maximum increase of 10- to 20-fold by 6 to 12 h, and was still evident after 3 d in culture. The distribution of NGF mRNA in different areas of the iris does not change during this time. This rapid increase in NGF mRNA is also seen in the fully innervated iris in vivo after trauma to the anterior chamber. In contrast, denervation to varying degrees in situ had no effect on NGF mRNA levels. Neither removal of sympathetic innervation by surgical or chemical methods nor combined surgical removal of sympathetic and sensory innervation detectably altered NGF mRNA content. Thus, denervation of the rat iris in situ does not cause the observed accumulation of NGF by increasing the level of NGF mRNA, and the increase in NGF content must be due to other factors.     

The effects of denervation on protein turnover of the soleus and extensor digitorum longus muscles of adult mice.

1. Changes in protein turnover of the soleus and EDL muscles of adult mice have been studied 1, 7 and 80 days after denervation. 2. Increased rates of protein degradation 7 and 80 days post-denervation correlated with the atrophy and loss of protein from these muscles. 3. Rates of protein synthesis in the EDL decreased 24 hr after nerve section. However, these synthetic rates increased again to become higher in the 7 day denervated muscles compared with their controls. These latter anabolic changes are inconsistent with the concept of a denervated muscle being inactive. 4. These findings have been compared with a similar study on muscles of growing rats. Any passive stretching of the denervated muscles by continued bone growth appears unlikely to be a crucial factor explaining the increased rates of protein synthesis 7 days after denervation.     

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.