Austin J. MacInnis

DNA from anaerobic adult Ascaris lumbricoides and Hymenolepis diminuta mitochondria isolated by zonal centrifugation.

Abstract 1. Reorienting gradient zonal centrifugation was used to isolate large quantities of mitochondria from the parasitic helminths, Ascaris lumbricoides and Hymenolepis diminuta. This technique may be particularly useful in obtaining mitochondria or other organelles present in small quantities from any tissue. 2. Electron micrographs of mitochondrial DNA released from osmotically shocked mitochondria and from purified mitochondrial DNA revealed a circular molecule with an average contour length of 4.76 ± 0.03 μ m in H. diminuta and 4.79 ± 0.03 μ m in A. lumbricoides. 3. Mitochondrial DNA was subsequently isolated from both helminths and characterized using equilibrium centrifugation in CsCl. The buoyant density of mitochondrial DNA was 1.690 g/cm3 in A. lumbricoides and 1.691 g/cm3 in H. diminuta. Estimates made from buoyant density measurements and thermal transition values indicated a base composition of 31 % G + C for A. lumbricoides and 32 % for H. diminuta mitochondrial DNA.

Rapid detection of malaria and other bloodstream parasites by fluorescence microscopy with 4'6 diamidino-2-phenylindole (DAPI).

DAPI is a fluorescent dye which appears to complex specifically with DNA. We have used this probe to detect and identify malarial infections by fluorescence microscopy. Experiments were conducted using Plasmodium berghei yoeli--infected mouse blood, P. lophurae--infected duck blood, and P. vivax--infected human blood. Infected avian blood was used to detect parasites within nucleated erythrocytes. Control blood smears from uninfected hosts revealed fluorescence only in the leukocytes of mammalian blood or in nuclei of leukocytes and erythrocytes of avian blood. Cytoplasmic staining of red blood cells was absent in all controls. In contrast, the cytoplasm of infected red blood cells was stippled with fluorescence centers. Ring forms, trophozoites, segmenters, and merozoites frequently were observed. This simple procedure can be applied directly to routine clinical analysis, as well as experimental procedures, DAPI can also be used to stain other parasites, including nuclei in microfilariae.

Physiological relationships and circadian periodicities in rodent trypanosomes

Trypanosome circadian rhythms in rats infected with Trypanosoma lewisi and mice infected with T. duttoni (equals T. musculi) were observed. Peak numbers of trypanosomes were recorded at nightfall and fewest organisms seen at daybreak. Reversal of the photoperiod resulted in a comparable reversal of the trypanosome parasitaemia. Periodicities of blood glucose levels and circulating leucocytes were relatively similar in T. lewisi-infected rats to rhythms previously defined in normal aimals. In trypanosome-infected mice, circulating leucocytes had a peak at 1800 hours and were minimal at midnight. Immune serum and epinephrine apparently had opposite effects on numbers of circulating rat trypanosomes; antisera reduced and epinephrine increased the numbers. Increased motor activity appeared to induce increased parasitaemia. Results of these and other studies suggest that in diurnally active hosts, trypanosome periodicities are characterized by increasing numbers in the circulation throughout the day and reach a peak just before darkness. In nocturnally active animals a similar rhythm was observed with only a slight phase change; circulating trypanosomes increased throughout the day and were maximal soon after nocturnal onset.

The in vitro effects of farnesol and derivatives on Hymenolepis diminuta.

Employing an in vitro maintenance system, in which 8-day-old Hymenolepis diminuta survives for 24 hr (Fioravanti and MacInnis, 1976), it was found that farnesol or farnesal supplementation of the medium had no beneficial effects on maintenance and these substances induced necrosis at higher concentrations. Similar experiments utilizing Schillers (1965) culture system demonstrated that neither farnesol, farnesal, nor farnesyl methyl ether exhibited growth promoting effects and were toxic to the worms at higher concentrations. In addition, neither the 2-cis, 6-trans nor the 2-trans, 6-trans-isomers of farnesol promoted growth in the Schiller system and at higher concentrations resulted in severe necrosis within 24 hr.

Characterization and hybridization of DNAs of gyrocotylidean parasites of chimaeroid fishes.

Abstract DNA was isolated from four species of Gyrocotyle and its base composition determined. DNA extracted from alcohol-preserved worms was suitable for some physical-chemical characterization, and for in vitro DNA hybridization studies. Although Gyrocotyle fimbriata readily accumulated adenine, thymine or thymidine, no detectable incorporation into nucleic acids was observed during a 24-h period. Thus, to prepare radioactive DNA for DNA-DNA heteroduplex experiments, the isolated DNA was tritiated in vitro , Heteroduplex formation between DNA from Gyrocotyle rugosa and Gyrocotyle fimbriata was much greater than the amount of hybridization of DNA from either of these species with that from Gyrocotyle maxima or Gyrocotyle parvispinosa . These results, although preliminary, suggest that the technique of DNA hybridization might be developed as a useful method in parasite taxonomy.

ISOLATION OF POLY A(+) RNA FROM SCHISTOSOMA MANSONI AND IMMUNOPRECIPITATION OF ITS IN VITRO TRANSLATION PRODUCTS

Total RNA was extracted from S. mansoni by homogenization in 4 M guanidine thiocyanate followed by centrifugation through cesium chloride. Poly A(+) RNA was isolated by oligo(dT)-cellulose chromatography. The recovered poly A(+) RNA was subsequently shown to stimulate protein synthesis in a cell-free, rabbit reticulocyte system. Among the translation products were polypeptides with molecular weights of 18, 20, 26, 39, 42, and 50 kilodaltons that were recognized by rabbit anti-schistosome-denuded body IgG. Human infection serum IgG precipitated polypeptides with molecular weights of 16, 18, 30, 35, 37, and 42 kilodaltons. These results identify several mRNAs that may provide useful templates for preparation of cDNA for eventual cloning or as probes in various related experiments.

EFFECTS OF HYCANTHONE AND PRAZIQUANTEL ON MONOAMINE OXIDASE AND CHOLINESTERASES IN SCHISTOSOMA MANSONI

The effects of hycanthone and praziquantel on the activities of monoamine oxidases and cholinesterases were studied in the 600-g supernatant from homogenates of Schistosoma mansoni and mouse liver or brain. Hycanthone was shown to be a very potent inhibitor of monoamine oxidases from worms and mouse liver. Hycanthone also inhibited the specific and nonspecific cholinesterases of S. mansoni, but cholinesterase from mouse brain was not affected significantly by this drug. Praziquantel showed no effect on monoamine oxidase from mouse liver or the parasite; however, it was slightly inhibitory to S. mansoni cholinesterases at very high concentrations. Mouse brain cholinesterase required an even higher concentration of praziquantel to observe inhibition. The inhibition of monoamine oxidase in S. mansoni by hycanthone adds a new mode of action to our knowledge of this compound, and suggests another possibility for the development of future anthelminthics.

Metabolic indices for evaluating the in vitro maintenance of Hymenolepis diminuta in the presence and absence of various additives.

An in vitro maintenance system for H. diminuta was devised by modifying the cultivation procedure of Schiller (1965). In this diphasic maintenance system, tissue culture medium (Triple Eagles or NCTC-135) was used, in lieu of whole blood, as the 30% supplement to the agar phase. This provides a more defined system suitable for studying the effects of various additives. Morphological criteria were established which aided in assessing the efficacy of media used for the maintenance of 6- and 8-day-old H. diminuta. When successfully maintained, worms exhibited an intact scolex and neck region, undulatory movements along the strobila as well as integumentary and strobilar integrity. A more sensitive method for evaluating the maintenance of 8-day-old worms employed metabolic indices. Wet weight, protein and glycogen levels for 8-day-old H. diminuta served as base-line data allowing estimation of protein and glycogen contents of each worm prior to maintenance. Following maintenance, ratio of final to initial protein and final to initial glycogen levels (metabolic indices). A metabolic index approaching or exceeding unity suggested a reasonably intact metabolism. The addition of sodium taurocholate to the maintenance media appeared beneficial to the worms by prolonging the retention of normal signs. A combination of additives, taurocholate-nucleosides-lipids, improved the maintenance of H. diminuta for periods exceeding 24 hr as determined by observational criteria and metabolic indices. However, addition of a lipid mixture, or a lipid mixture prepared with a low concentration of taurocholate was not beneficial over a 24 hr period. The maintenance system, observational criteria, base-line data and metabolic indices should be useful for future in vitro studies requiring long-term incubation.

High-resolution localization of drug binding sites

Previous attempts at the ultrastructural identification of drug binding sites have been thwarted by denaturation and extraction of the tissue, and unreliable specificity of the localization procedure. We minimized the first problem by using the low denaturation method reported by Sjostrand and Barajas (1968, J. Ultrastruct. Res. 25, 121). Specific and reliable localization of lucanthone and methylene blue in the Ehrlich Ascites Circinoma was accomplished by precipitation of the drugs with phosphotungstic acid a pH 7. Lucanthone was localized only in the heterochromatin and interchromatinic granules, which supports the biochemical reports of lucanthone binding to DNA. The useful light microscopic stain, methylene blue was localized also in nuclear locations such as: the nucleolus, heterochromatin, and interchromatinic granules. This new approach should have a wide application in the analysis of the subcellular distribution, toxicology, structure-activity relationships, and possible mechanism of action of various drugs.

Development of a cell-free protein-synthesizing system from Schistosoma mansoni and a comparison of the effects of hycanthone and praziquantel on this system.

A cell-free, protein-synthesizing system was developed using polysomes and pH 5 fractions isolated from Schistosoma mansoni. Under optimal conditions, the system incorporated 35-S methionine into TCA-precipitable protein at a rate of approximately 1.8 pmoles/hr/microgram polysomal RNA. Praziquantel had no significant effect on the rate of protein synthesis, whereas hycanthone, at concentrations of 0.125 and 0.5 mM, caused a 136% and 515% stimulation, respectively. The mechanism of the stimulation is unknown. This S. mansoni--cell-free system for protein synthesis has been standardized so that new drugs can now be screened with relative ease for their effects on in vitro protein synthesis or charging of tRNA.