Freddie L. Riley

Isolation and Characterization of DNA from Kinetoplasts of Leishmania enriettii

The DNA of Leishmania enriettii can be separated by equilibrium sedimentation in cesium chloride into a major band of density 1.721 and a minor component of density 1.699. DNA from isolated kinetoplasts of this protozoan was identified as the less dense minor component.

Comparison of the DNA's obtained from brain nuclei and mitochondria of mice and from the nuclei and kinetoplasts of Leishmania enriettii

Abstract DNA has been isolated from mouse-brain-mitochondrial preparations, free of nuclear contamination. After purification, the buoyant density and thermal denaturation profiles of this DNA have been compared with those of mouse nuclear DNA, and with the nuclear and kinetoplast DNA of Leishmania enriettii . The buoyant density of mouse mitochondrial DNA was 1.701 and its T m 82.8°. The C-G content was 42 %, calculated from the density, and 33 %, calculated from its T m . The buoyant density of mouse nuclear DNA was 1.702, and its T m 86.2°, corresponding to a C-G content of 43 % and 41 %, respectively. The buoyant density of the Leishmania kinetoplast DNA was 1.702, and its T m 83.0°, corresponding to a G-C content of 43 % and 33 %, respectively. The buoyant density of the Leishmania nuclear DNA was 1.721 and its T m 92.8°, corresponding to a C-G content of 62 % and 57 %, respectively. On thermal denaturation, the initial hyperchromicity of the two nuclear DNAs was approx. 25 %, that of mitochondrial DNA about 29 %, and that of kinetoplast DNA about 8 %. The degree of renaturation of mitochondrial DNA was 21.5 %, that of kinetoplast DNA 78.6 %. Mouse-nuclear DNA renatured 10.3 %, but Leishmania nuclear DNA 36.4 %. From these and other data it was concluded that the DNA from the protozoal kinetoplast was more homogeneous than protozoal nuclear DNA. Both protozoal DNAs appeared more homogeneous than mouse-mitochondrial DNA, whereas mouse nuclear DNA was the most heterogeneous. The data are consistent with the theory regarding the existence of mitochondrial inheritance.

IMMUNE MODULATING EFFECTS OF POLY ICLC

This presentation will compare immune modulation brought about by interferon with those brought about by interferon inducers, particularly doublestranded RNA. If the only effect of the interferon inducer was the induction of interferon, then we would expect that interferon and its inducers would have the same effect in any given immune phenomenon, be it no effect, enhancement or inhibition. The fact that inducers can elicit a higher level of circulating interferon in vivo than is readily obtainable by administering exogenous interferon might modify this expectation in that large amounts of interferon might act differently from small amounts of interferon. However, within this limitation, interferon inducers might be expected to act like interferon. Indeed, there are many instances where this is the case. You will hear of them during this session. One such is the enhancement of natural killer cell activity by interferon and by poly

The Mechanism of the Antitumor Action of Poly I: Poly C

We have already reported RNA, po1yinosinic:polycytidylic acid, (poly 1:poly C) exerts inhibitory action on a number of experimental tumors i n animals. were of three types; (1) a decrease i n growth ra te o f the tumor when treatment was s ta r ted a few days a f t e r implantation of the tumor, (2 ) regression of already well established tumors, (3) increased survival time i n the animals bearing the tumors. Table 1 gives a l i s t of most of the tumors examined. In addition, w i t h Dr. Herman duBuy, we have examined a melanoma, the Bittner mamnary tumor, the Ehrl i ch asci t es tumor, and have found comparable resul ts .

The effect of polyinosinic,polycytidylic acid on tumor metabolism.

Summary The synthetic double-stranded RNA, polyinosinic: polycytidylic acid (poly I:poly C), strongly inhibits in vivo RNA and protein synthesis in three different tumors in mice. Macromolecule synthesis in normal organs of these mice may be inhibited or enhanced, depending to some extent on the strain of mouse. Administration of poly I:poly C to the mice also leads to alterations in the specific radioactivity of acid-soluble precursor pools, sometimes increasing and sometimes decreasing such specific activity. There was no discernible relationship between the nature of the alteration in precursor specific activity and the direction of effect on macro-molecule synthesis.

Tetracycline Fluorescence in Permeability Studies of Membranes around Intracellular Parasites

Certain protozoa, bacteria, and viruses when phagocytosed by host cells become surrounded by an intracytoplasmic boundary. This membrane prevents the fluorescent antibiotic tetracycline from entering the parasites when it is added to the medium, since they show no fluorescence, whereas extracellular parasites are immediately visible. As soon as the host cell dies, the intracellular parasites also become visible. This indicates that the boundary probably is of host origin. This phenomenon provides a means for selective permeability studies of such boundaries. A similar exclusion of tetracycline from certain extracellular parasites is seen in the presence of whole serum.

Studies on the mechanism of interferon action: Characterization of polysome-associated cellular RNAs modified by interferon

Abstract A recent publication indicated that certain polysome-associated RNA species are altered in interferon-treated cells. The present data show that these RNA species are poly(A)-containing mRNAs, RNAs without a poly(A)-rich region and tRNAs. In addition, we show that in polyacrylamide gels in aqueous medium as well as in nonaqueous medium (formamide) the mRNAs from interferon-treated cells migrate more slowly than do control cell mRNAs, suggesting that the interferon mRNAs are slightly larger than normal. Transfer RNAs from interferon-treated cells, on the other hand, move more slowly than control tRNAs in aqueous medium, but not in formamide, suggesting that the difference in mobility in tRNAs is associated with factors other than size.

Polyriboinosinic-Polyribocytidylic Acid-Poly-L-lysine Complex [Poly(ICL)] without Carboxymethylcellulose (CMC): A New Primate-Effective Interferon Inducer

Abstract The complex of polyriboinosinic-polyribocytidylic (poly(I)·poly(C)) with poly-L-lysine in 0.5% carboxymethylcellulose (CMC) [poly(ICLC)] has proven to be an effective interferon inducer in primates, including man. Since no mechanism is known by which the body can degrade CMC, a new complex of lower molecular weight, which contains poly I·poly C complexed with poly-L-lysine [poly(ICL)] but without CMC, was developed. This compound is slightly more resistant than poly(ICLC) to hydrolysis by RNase-A and is also an effective inducer of interferon in nonhuman primates. The new compound without CMC is also less toxic in mice than is poly(ICLC) as indicated by LD50.

Utilization of Stabilized Forms of Polynucleotides

Within a few years after the description of interferon by Isaacs and Lindenmann (1957), it was recognized that interferon potentially was a broad-spectrum antiviral agent of possibly high value in clinical medicine. However, the difficulty of preparing enough interferon, either nonhuman or human, prevented the adequate testing of this potentiality. Serious efforts were made to find nonreplicating agents that would cause the host to synthesize its own interferon in large quantity. While a number of compounds were found that are capable of causing mice, and possibly humans, to make interferon, they either induced too small amounts or were too toxic (Merigan 1973). Field et al. (1967) reported that a number of natural and synthetic double-stranded (ds) RNAs are capable of inducing interferon. In particular, the dsRNA polyinosinic-polycytidylic acid (poly(I)-poly(C)) was highly effective in rodents as an interferon inducer (Field et al. 1967), as an antiviral agent (Parks and Baron 1968; Worthington et al. 1973) and as an antitumor agent (Zelezinck and Bhuyan 1969; Levy et al. 1969).