Evidence for the viral nature of the lactic dehydrogenase (LDH) agent, discovered in the plasma of m a n y tumor-bearing mice by Riley (1), has been obtained by several laboratories (2, 3). The ability of this agent to cause an increased activity of a number of enzymes in mouse plasma (4), persisting during the lifetime of the host (1) without causing apparent cellular damage merits further study.This paper will present data on maintenance, titration, and partial purification of the L D H agent, its resistance toward organic solvents, its morphology and size as determined by electron microscopy, its activity as an interferon producer, and its lack of antigenicity. Materials and MethodsStrain of LDH Agent.--The strain was obtained from a naturally infected Ehrlich ascites carcinoma. This Ehrlich ascites strain had been maintained for 2 years in CDF1 mice (NIH strain).Infectivity Titer.--Infectivity of the agent was assayed by injecting 0.1 ml of 10-fold serial dilutions, made in Eagle's medium plus 20 per cent veal infusion broth (5), into NIH G-P Swiss (20 to 25 gin) mice, 5 mice per dilution. Four to 5 days after inoculation blood was collected by orbital bleeding (6) with capillary pipettes, wetted with a heparin solution containing 1000 U.S.P. units heparin per ml, and pooled. LDH ~ctivity was determined on the plasma obtained from each pool by a modification of the method of Wroblewski and Ln Due (7). The number of infected mice at each dilution was estimated from a scale representing the stepwise increase of LDH activity contributed by each additional infected mouse (m~/e infra) and the results calculated by the Karber method. The titer was expressed as the negative logx0 dilution of the sample which induced an increased lactic dehydrogenase activity in 50 per cent of the mice (IDa0 per ml).Preparation of LDH Agent.--Blood plasma or ascitic fluid from Ehrlich ascitic carcinomabearing mice (v/de infra), infected 24 hours previously with the LDH agent, was harvested and centrifuged at 900 Rcz for 10 minutes. The supernatant was diluted with an equal volume of 0.3 M potassium citrate and 0.003 per cent hyaluronidase, and digested at room temperature for 1 hour (8). It was centrifuged at 4000 Rc]v for 20 minutes.
The existence of extranuclear hereditary factors implies the presence of an extranuclear nucleic acid, to a certain degree different from nuclear DNA.' The presence of DNA in mitochondria, plastids, and kinetoplasts with melting profiles, reannealing properties, and buoyant densities different from those of the corresponding nuclear DNA's has by now been demonstrated by various groups (e.g., see refs. 1-7). There are only a few reports regarding hybridization reactions between DNA from extranuclear sites with the corresponding nuclear DNA. Shipp et al.,5 using tobaccochloroplast DNA and nuclear DNA of matured tobacco leaves, found 10-15 per cent binding of P-labeled nuclear DNA fragments and 0.6 per cent binding of P-labeled chloroplast DNA fragments to nuclear DNA embedded in agar, indicating no detectable homology. Dawid9 reported that frog-egg DNA fragments, apparently of mitochondrial origin,10 (at the concentrations used) do not compete with C14-labeled tadpole DNA in their reaction with homologous single-stranded frog DNA embedded in agar. He further showed in hybridization experiments that egg complementary RNA hybridized to a small degree with erythrocyte or liver nuclear DNA. The present paper will present data on hybridization reactions between nuclear and kinetoplast DNA's of Leishmania enriettii, and nuclear and mitochondrial DNA's from mouse liver. Materials and Methods.-Preparation of Leishmania DNA's: Kinetoplast and nuclear DNA of Leishmania were prepared by methods described previously.7 Each batch of kinetoplast DNA was characterized by the formation of only one rapidly appearing band following density gradient centrifugation in CsCl (d = 1.699, 44,770 rpm, 18 hr).11 C'4-labeled Leishmania nuclear DNA was obtained by the addition of C14-thymidine (100 yAe in 0.8 mg) to two Erlenmeyer flasks each containing 40 ml Senekjie medium, and inoculated with ca. 1.2 X 107 organisms each. The organisms were harvested after 3 days, filtered through cheesecloth, and centrifuged, and the pellet was washed once with water. The DNA was then prepared as previously described,7 and fragmented by mechanical shear of 7.0-10.5 X 106 gm/cm2 to a molecular weight of 3-5 X 101 (ref. 12). Preparation of mouse-liver DNA's: Mouse mitochondrial DNA was prepared as described before,'1 with some modifications. The DNase treatment was eliminated. The final pellets were further purified by washing with saline, and centrifuged at 18,000 g for 20 min, and the bottom layer was discarded. After addition of the solution containing Tris, sodium lauryl sulfate, and pronase, as described before. the pellets were digested. The preparation was twice extracted with phenol, each time followed by centrifugation in a Spinco ultracentrifuge in a SW25 rotor at 16,000 rpm for 20 min.3 The bottom layer and the interphase were discarded. Further purification proceeded as described before, except that the preparation was sheared at 7.0-10.5 X 106 gm/cm2 before it was placed on the Sephadex column. Each batch of mitochondrial DNA was characterized ...
The property of tetracycline to induce fluorescence has been used to determine its localization in living cells. It was found that this antibiotic, as well as the related antibiotics oxytetracycline and chlorotetracycline, specifically combines with the mitochondria of living cells, either in tissue culture or in fresh preparations from various organs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.