John F. Roberts

Isolation of bodies containing the cyanide-insensitive glycerophosphate oxidase of Trypanosoma equiperdum

Abstract 1. 1. Differential mitochondrial fractions collected from Trypanosoma conorhini homogenates exhibited glycerophosphate oxidase (GPO) activities that were inhibited by cyanide, but mitochondrial fractions from T. equiperdum contained a cyanide-insensitive GPO. 2. 2. Gel-filtration chromatography and disc electrophoresis separated T. conorhini sodium dodecylsulfate-soluble mitochondrial proteins into twelve fractions and T. equiperdum into five fractions. 3. 3. Sucrose density-gradient centrifugation of the T. equiperdum mitochondrial fractions resolved two sub-fractions: a “heavy” DNA-containing fraction and a “light” GPO-containing fraction. 4. 4. Electron micrographic inspecteion of the “light” fraction showed it was composed of flagella and spherical bodies that are abundant in teh cytoplasm of T. equiperdum .

Trypanosoma brucei: recognition in vitro of two developmental forms by murine macrophages.

Abstract In vitro , murine macrophages attach to the midgut form of Trypanosoma brucei in the absence of exogenous proteins. Recognition appears to be specific and saturable. Attachment is mediated by a non-Fc, non-C3 receptor on the macrophage plasma membrane. The attachment mechanism is protease sensitive, temperature sensitive, requires the presence of divalent cations, and is functional in fetal calf serum-free medium. The midgut form is also lysed in normal rabbit serum by activating the alternative complement pathway. The form isolated from the blood is neither lysed in normal serum nor is it spontaneously recognized by the macrophage, in vitro . Partial trypsinization of the bloodstream form, however, results in both the triggering of alternative complement pathway lysis and spontaneous uptake into macrophages. Murine macrophages attach to the midgut form of T. brucei by a receptor on the macrophage plasma membrane which is capable of recognizing particulate activators of the alternative complement pathway.

Regulation of oxidase enzyme systems in trypanosomes.

Abstract 1. 1. QO2, succinoxidase and glycerophosphate oxidase (GPO) levels (0·533, 0·367 and 0·334 μmoles O2 hr−1 mg protein−1) were determined for Trypanosoma conorhini. 2. 2. Kinetoplastic and dyskinetoplastic T. equiperdum had higher QO2 (2·51 and 1·87) and GPO (2·38 and 1·15) levels but lacked measurable succinoxidase. 3. 3. Reduced O2 availability during growth of T. conorhini gave reduced succinoxidase and increased GPO activities. 4. 4. Acriflavin and chloramphenicol added at initiation of cultures of T. conorhini gave decreased growth and QO2 while only acriflavin decreased succinate ferricyanide reductase activity. 5. 5. Acriflavin added to exponentially growing T. conorhini reduced only succinate ferricyanide reductase activity and increased cyanide-insensitive QO2 and GPO levels.

Microbodies inCrithidia fasciculata

SummaryElectron microscopy of thin sections ofCrithidia fasciculata showed cytoplasmic organelles 0.2–0.8 μm in diameter containing a finely granular matrix limited by a single membrane. Utilization of cytochemical techniques (DAB) for endogenous catalase revealed pronounced deposition of a highly electron-opaque material in these organelles. Reaction of cells with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole. It is concluded that this organelle is a microbody on the basis of cytological and cytochemical similarities with microbodies from plant and other animal cells.

Activities and isozymes of malate and lactate dehydrogenases in culture and bloodstream form trypanosomes.

Abstract 1. 1. Lactate dehydrogenase (LDH) of the culture from Trypanosoma conorhini has an activity of 158 units/min per mg protein and is composed of five isozymes. This LDH had abnormal Michaelis-Menten kinetics and is denatured by 2 M urea. 2. 2. LDH activities of the kinetoplastic and dyskinetoplastic bloodstream forms of T. equiperdum are 23 and 33 units/min per mg protein, respectively. LDHs of both forms are composed of two isozymes. 3. 3. The malate dehydrogenase (MDH) activity of T. conorhini is 7337 units/min per mg protein. The MDH is composed of two isozymes when NAD is used and three isozymes when NADP is used as the cofactor. The anodal isozyme was the mitochondrial MDH and the soluble fraction contained both MDH isozymes. 4. 4. The MDH activity of the kinetoplastic and dyskinetoplastic forms of T. equiperdum are, respectively, 355 and 298 units/min per mg protein. The MDH of both forms was composed of three closely spaced cathodal isozymes. 5. 5. LDH increased in activity as the T. conorhini cultures aged. MDH increased and LDH decreased in cultures with a larger surface to volume ratio than the cultures normally used.

In vivo evaluation of reuterin and its combinations with suramin, melarsoprol, dl-α-difluoromethylornithine and bleomycin in mice infected with Trypanosoma brucei brucei

1. Trypanosoma brucei brucei-infected mouse models treated with a new antibiotic, reuterin, showed reduction of the levels of parasitemia and prolonged survival of the mice. 2. Cures of the parasitemia were observed in groups of mice treated with combinations of reuterin and suramin or melarsoprol. 3. Reuterin administered in combination with bleomycin or DL-alpha-difluoromethylornithine showed temporary remission of the parasitemia in groups of mice.

The in vitro efficacy of reuterin on the culture and bloodstream forms of Trypanosoma brucei brucei.

1. The in vitro effects of a new antibiotic, reuterin, were determined for culture and bloodstream forms of Trypanosoma brucei brucei. It inhibited growth of the culture forms and motility, viability and DNA and protein syntheses of culture and bloodstream forms. 2. Reuterin administered with inhibitors of ribonucleotide reductase (hydroxyurea and deoxyadenosine) was synergistic only for growth of the culture form of the parasite. 3. Reuterin was trypanocidal at lower doses than DFMO, Mel B, and Suramin and was antagonistic when used with these drugs.

Adaptation of Crithidia fasciculata to growth in the presence of carbonyl cyanide m-chlorophenylhydrazone☆

Abstract 1. Crithidia fasciculata will grow in medium containing 10 −5 M carbonyl cyanide m -chlophenyldrazone (CCCP) after a prolonged lag period of 75 hr. 2. The resulting cells are resistant to the uncoupler and upon subinoculation exhibit growth characteristics similar to those of normal cultures. 3. The adapted cells lose their resistance to CCCP when cultured for 30 hr medium. The adapted cells do not lose their resistance when held for similar periods of time in medium that does not support cell division. 4. Adapted cells are smaller than control cells and they tend to swim in circular paths. 5. In the absence of exogenous substrate, normal and adapted cells utilize oxygen at apporimately the same rate. However, the respiration of adapted cells is not markedly stimulatd upon addition of glucose or proline. 6. Both cell types were equally sensitive to the respiratory chain inhibitors KCN and diphenylamine. The respiration of CCCP-adapted cells was more sensitive than that of control cells to CCCP and 2,4-dinitrophenol.

A comparative study of the ribonucleic acids of three species of trypanosomatids.

Abstract 1. 1. The ribosomal RNAs of Crithidia fasciculata, Leishmania tropica, and Trypanosoma brucei have sedimentation values of 25–27S, 18–20S and 5S. 2. 2. The molecular sizes of the two heavier components are 1.24–1.28 M (106 daltons) and 0.84–0.89 M as determined by polyacrylamide gel electrophoresis. 3. 3. The RNA of the three species studied shows a tendency to interact forming a complex that is very difficult to separate by sucrose gradient centrifugation. 4. 4. The 25–27S RNA is very unstable. 5. 5. The 18–20S RNA has a molecular size higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba.

In vitro effect of aflatoxin b1 on rat liver macrophages (kuffer cells)

The effect of aflatoxin B1 on the uptake and incorporation of [3H]leucine and [3H]uridine and on phagocytosis of latex particles was studied using cultures of rat liver macrophages (Kuffer cells). Aflatoxin B1 inhibited the incorporation of both isotopes, but inhibition of uridine incorporation was greater than that of leucine, suggesting that RNA synthesis was a major site of inhibition. Aflatoxin B1 also inhibited phagocytosis of latex particles in a time- and dose-dependent manner.