Regulation of the activity of caspases by L-carnitine and palmitoylcarnitine.

L-Carnitine facilitates the transport of fatty acids into the mitochondrial matrix where they are used for energy production. Recent studies have shown that L-carnitine is capable of protecting the heart against ischemia/reperfusion injury and has beneficial effects against Alzheimer's disease and AIDS. The mechanism of action, however, is not yet understood. In the present study, we found that in Jurkat cells, L-carnitine inhibited apoptosis induced by Fas ligation. In addition, 5 mM carnitine potently inhibited the activity of recombinant caspases 3, 7 and 8, whereas its long-chain fatty acid derivative palmitoylcarnitine stimulated the activity of all the caspases. Palmitoylcarnitine reversed the inhibition mediated by carnitine. Levels of carnitine and palmitoyl-CoA decreased significantly during Fas-mediated apoptosis, while palmitoylcarnitine formation increased. These alterations may be due to inactivation of beta-oxidation or to an increase in the activity of the enzyme that converts carnitine to palmitoylcarnitine, carnitine palmitoyltransferase I (CPT I). In support of the latter possibility, fibroblasts deficient in CPT I activity were relatively resistant to staurosporine-induced apoptosis. These observations suggest that caspase activity may be regulated in part by the balance of carnitine and palmitoylcarnitine.

A Trypanosoma brucei bloodstream form mutant deficient in ornithine decarboxylase can protect against wild-type infection in mice.

A Trypanosoma brucei bloodstream mutant in which both copies of the ornithine decarboxylase (ODC) gene were knocked out (ODC mutant) was used to determine the biological functions of ODC in T. brucei. Growth of the mutant cells ceased within 12-24 h in regular culture medium deficient in polyamines, but could be rescued by supplementation with 1 mM putrescine. A mouse model of T. brucei infection was used to determine whether the mutant was still infective and was found to develop either extremely low or undetectable levels of parasitemia, suggesting that in T. brucei, ODC activity is essential for establishing an infection. Furthermore, when these mice were subsequently challenged with wild-type T. brucei cells expressing the same variant surface glycoprotein (VSG), they did not develop any parasitemia, indicating that inoculating the mice with the attenuated ODC mutant had conferred protection against challenge by wild-type cells. These results were reproduced in C57BL/6J mice deficient in alpha-beta and gamma-delta T-cell receptors. However, no protection was observed in rag-2 knockout mice deficient in both B and T lymphocytes or in C57BL/10J mice deficient only in B lymphocytes. The results thus suggest that the ODC mutant could induce a T-lymphocyte-independent but B-lymphocyte-dependent immunity against wild-type cells of the same VSG. Such a mechanism of immunity has been elicited only by live T. brucei cells, but not by isolated VSGs or radiation-killed trypanosomes. This ODC mutant may thus represent a genuinely attenuated T. brucei bloodstream form capable of immunizing mammals against infections by African trypanosomes of the same VSG subtype without causing detectable infection by itself. The observation also raises the interesting likelihood that the in vivo treatment of T. brucei bloodstream forms with alpha-DL-difluoromethylornithine is a de facto attenuation of the parasitic organisms, which may very well result in B-lymphocyte-dependent host immune responses to subsequent infections by parasites of the same VSG subtypes.

The role of proteolysis during differentiation of Trypanosoma brucei from the bloodstream to the procyclic form.

The in vitro differentiation of Trypanosoma brucei from bloodstream to procyclic (insect) forms is accompanied by diminishing variant surface glycoprotein (VSG) and increasing levels of procyclin and phosphoenolpyruvate carboxykinase (PEPCK). In this study, we examined the fate of several glycolytic enzymes of T. brucei during differentiation. We observed a down-regulation of glycosomal phosphoglycerate kinase (gPGK) during differentiation. In contrast, intracellular levels of glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), aldolase (ALD), and phosphoglucoisomerase (PGI) remained unchanged during differentiation and apparently continued to be synthesized in the procyclic form. To determine the potential role of proteasomes and other proteases during the differentiation process, we tested the effect of lactacystin, a specific inhibitor of proteasome activity, and morpholinourea-Phe-homoPhe-benz-alpha-pyrone (P27), a selective inhibitor of cysteine proteases, on the in vitro differentiation of T. brucei. Cells differentiated normally in the presence of 1 microM lactacystin, which confirmed our previous observation that this differentiation does not require crossing any phase boundaries in the cell cycle (Mutomba and Wang, Mol Biochem Parasitol 1996;80:89-102). But the cells thus differentiated did not increase in number and retained gPGK. Cells differentiated under 2 microM P27 also proceeded at a normal rate but failed to multiply and retained gPGK. However, most of the differentiated cells under 2 microM P27 also retained VSG on the cell membrane surface and expressed higher levels of procyclin suggesting that a cysteine protease(s) may be involved in releasing VSG and partially reducing procyclin during differentiation. This cysteine protease(s) has been tentatively identified in the procyclic cells as a 48 kDa protein through labeling of cysteine protease(s) with a biotinylated P27 homolog K02 (morpholinourea-Phe-homoPhe-vinylsulfone).

Inhibition of proteasome activity blocks cell cycle progression at specific phase boundaries in African trypanosomes.

Proteasomes are one of the cellular complexes controlling protein degradation from archaebacteria to mammalian cells. We recently purified and characterized the catalytic core of the proteasome, the 20S form, from Trypanosoma brucei, a flagellated protozoa which causes African trypanosomiasis. To identify the role of proteasomes in African trypanosomes, we used lactacystin, a specific inhibitor of proteasome activity. Lactacystin showed potent inhibition of the activity of 20S proteasomes purified from both bloodstream and procyclic (insect) forms of T. brucei (IC50 = 1 microM). It also inhibited proliferation of T. brucei cells in culture assays, with 1 microM inhibiting growth of bloodstream forms, whereas 5 microM was required to block proliferation of procyclic forms. Analysis of the DNA content of these cells by flow cytometry showed that 5 microM lactacystin arrested procyclic cells in the G2 + M phases of the cell cycle. Fluorescence microscopy revealed that most of the cells had one nucleus and one kinetoplast each, indicating that the cells had replicated their DNA, but failed to undergo mitosis. This suggests that transition from G2 to M phase was blocked. On the other hand, incubation of bloodstream forms with 1 microM lactacystin led to arrest of 30-35% of the cell population in G1 and 55-60% of the cells in G2, indicating that both transition from G1 to S and from G2 to M were blocked. These observations were also confirmed by using another inhibitor of proteasome, N-carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV), which arrested procyclic forms in G2, and bloodstream forms in both G1 and G2. These results suggest that proteasome activity is essential for driving cell cycle progression in T. brucei, and that proteasomes may control cellular functions differently in bloodstream and procyclic forms of T. brucei.

Effects of aphidicolin and hydroxyurea on the cell cycle and differentiation of Trypanosoma brucei bloodstream forms.

The effects of aphidicolin (APH) and hydroxyurea (HU) on the cell cycle and differentiation of Trypanosoma brucei bloodstream forms were studied. APH (0.1 microgram ml-1) inhibited cell division, but did not inhibit DNA synthesis. Most of the cells were arrested in the G2 phase of the cell cycle, with each cell containing two kinetoplasts, but only one nucleus. Recovery of the arrested cells showed a 24-h lag period compared to controls. Higher concentrations of APH (1 and 10 micrograms ml-1) were required to inhibit DNA synthesis, but the cells failed to resume growth after removal of the drug. Incubation of cells with HU (7.5 micrograms ml-1) did not inhibit DNA synthesis, but arrested cells after duplicating both the kinetoplast and the nucleus. Recovery from drug arrest also showed a 24-h lag period. We therefore conclude that neither APH nor HU arrests T. brucei at the G1/S phase boundary as anticipated. The mechanisms of cell cycle arrest by APH and HU are not through inhibition of DNA synthesis, but rather through unidentified pathways, leading to growth arrest prior to nuclear division and cytokinesis respectively. Since the arrested cells do not resume normal development immediately following drug removal, APH and HU should be regarded as unsuitable agents for synchronizing T. brucei bloodstream forms. T. brucei bloodstream forms arrested with either APH or HU differentiated normally into procyclic forms in vitro, indicating that a cycle of cell division is not required for initiation of differentiation, and that the process can be initiated and completed when cells are arrested at the G2/M and M/G1 phase boundaries.

Differentiation of a culture-adapted mutant bloodstream form of Trypanosoma brucei into the procyclic form results in growth arrest of the cells.

The bloodstream forms of Trypanosoma brucei monomorphic strain 427 serially passaged in rats can differentiate in vitro equally well in HMI-9, HMI-10, SDM-79 or Cunningham's medium into the insect (procyclic) forms by a simple temperature shift from 37 to 26 degrees C in the presence of citrate and cis-aconitate. The procyclic forms thus generated can also continue to multiply at 26 degrees C without replacing the culture medium. The same strain of T. brucei pre-adapted to grow as bloodstream forms in HMI-10 medium at 37 degrees C is also capable of differentiating showing a similar rate of variant surface glycoprotein (VSG) disappearance and appearance of phosphoenolpyruvate carboxykinase (PEPCK) under the same experimental conditions. However, appearance of both procyclin mRNA and procyclin protein is much delayed and the resulting procyclic forms cannot multiply. The culture-adapted bloodstream forms are capable of infecting rats, and the cells thus harvested from the rats can differentiate but cannot multiply in the same manner as the original culture-adapted bloodstream forms. Apparently, a certain variant has been selected during the adaptation of T. brucei bloodstream forms from rat blood to the culture medium. This variant could be a useful tool for identifying the genes involved in differentiation of T. brucei and multiplication of the procyclic forms. Comparison of the protein profiles between the wild-type and the variant during differentiation showed that a major protein band of about 70 kDa remained in the non-dividing variant procyclic forms but vanished in the rapidly dividing wild type procyclic forms. N-terminal determinations indicated that the 70-kDa protein band consists of bovine serum albumin and fetuin. Presumably these two serum proteins are actively taken up by the bloodstream forms via endocytosis. Since the procyclic forms are incapable of endocytosis, the serum proteins may be rapidly diluted in the growing wild type procyclic cells but remain unchanged in the non-dividing procyclic cells of the variant. Further studies are underway in trying to identify the key distinctions between these two lines of cells at the molecular level.