Polyamine Depletion Following Exposure to DL-α-Difluoromethylornithine Both In Vivo and In Vitro Initiates Morphological Alterations and Mitochondrial Activation in a Monomorphic Strain of Trypanosoma brucei brucei

ABSTRACT. DL-α-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), rapidly depletes cells of intracellular putrescine. When administered to animals and humans, DFMO cures acute infections of trypanosomiasis. In order to determine if the mechanism of drug action is related to initiation of transformation and biochemical alterations subsequent to polyamine depletion, trypanosome morphology and mitochondrial activation were studied in a monomorphic strain of Trypanosoma brucei brucei. Exposure of trypanosomes to DFMO in vivo in infected rodents or in vitro in culture resulted in a depletion of intracellular putrescine and a cessation of cell division without specific cytotoxicity. These events were followed by a transformation of the long slender bloodstream form to a short stumpy form via an intermediate morphology. Putrescine, the product of the ODC reaction, abrogates this effect. When introduced into SDM-79 medium, the intermediate form is capable of further transformation to an "insect" procyclic trypomastigote whereas the long slender form and short stumpy form are not. Short stumpy forms are incapable of binary fission and have lost their infectivity for the vertebrate host. In addition, the mitochondrial marker enzyme, NAD diaphorase, was found only in the short stumpy and intermediate forms. We hypothesize that the short stumpy phenotype may not be a viable stage in the natural transformation of the trypanosome from its mammalian host to the insect vector.     

Content,Synthesis, and Function of Polyamines in Trypanosomatids: Relationship to Chemotherapy1,2

Polyamines are multiply amine-substituted straight-chain aliphatics; their content in different tissues may vary widely, and their functions are many. Their main routes of biosynthesis originate from ornithine and methionine. Polyamine content and biosynthesis in tryposomatid flagellates are reviewed concluding with emphasis on their possible role as critical drug targets in these parasitic protozoa so pathogenic for man in large areas of the world.     

Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.     

Oxaloacetate Production via Carboxylations in Crithidia fasciculata Preparations

SYNOPSIS. Fractions containing soluble enzymes from Crithidia fasciculata had an ADP-linked phosphoenolpyruvate (PEP) carboxykinase. The enzyme produced ATP and oxaloacetate (OAA) from PEP, ADP and HCO⁻₃. OAA was determined as the endproduct of reactions by forming the 2,4-dinitrophenylhydrazone derivative; the hydrazone was identified by thin-layer chromatography. Approximate Michaelis constants (PEP, Mg, HCO⁻₃, ADP) were determined spectrophotometrically by linking OAA production to malic dehydrogenase. The PEP carboxykinase did not utilize GDP, UDP or IDP as cofactors; the metal requirement was also satisfied by Mn. The enzyme was inhibited by the biotin antagonists avidin and desthiobiotin.
A pyruvate carboxylase was also present in the preparations, generating OAA from pyruvate and ATP. The role of both enzymes in OAA production and subsequent production of succinate is discussed with regard to C. fasciculata and other trypanosomatids.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Effects of carbaryl and cyhexatin on survival and reproductive behaviour of Cydia pomonella (Lepidoptera: Tortricidae)

The effects of carbaryl, an insecticide and an abscission agent, and cyhexatin, an acaricide, on the survival, reproductive behaviour and subsequent egg hatch and larval migration of the codling moth, Cydia pomonella L., were investigated under laboratory conditions. Dilution series starting with the concentration recommended for high volume application in the field were evenly sprayed in plastic cups. Depending on the concentration used, both pesticides resulted in high adult mortality, males being more susceptible. Although the total number of the eggs laid by C. pomonella females was significantly reduced in most treatments, the females exposed to different residues of carbaryl laid significantly more eggs during the first 24 h of exposure. This was not the case for cyhexatin. While in all treatments almost 50% of the eggs were laid on the side of the cups, the spatial distribution of the eggs laid under the lid and on the floor was concentration dependent. With one exception, continuous exposure of the moths to carbaryl significantly reduced the number of spermatophores per female. Less than one spermatophore per female was transferred at the highest four concentrations of carbaryl and the highest three concentrations of cyhexatin; whereas, the number of spermatophores per female amounted to about four per female for the other treatments. In contrast to carbaryl, which exhibited a high ovicidal effect at all concentrations, the ovicidal activity of cyhexatin was low. This was manifested in a high rate of egg hatch and the extent of embryonic development in most of the treatments. Furthermore, while carbaryl killed the newly-hatched larvae at all concentrations, cyhexatin acted as a larvicide at the highest two concentrations only.