Lysis of malarial parasites and erythrocytes by ferriprotoporphyrin IX-chloroquine and the inhibition of this effect by proteins

Ferriprotoporphyrin IX(FP) lysed both erythrocytes and isolated Plasmodium falciparum as judged by decrease in turbidity of erythrocyte and parasite suspensions. The lytic effect of FP on erythrocytes was enhanced by chloroquine (CQ). In the presence of 2.5-20 microM CQ, 5 microM FP led to complete hemolysis within 45 min. However, the lytic effect of FP or FP-CQ on both erythrocytes and parasites was inhibited completely by proteins. The protein inhibition was non-specific. This finding, the failure of FP and FP--CQ to cause hemolysis and lysis of malarial parasites in a protein-containing medium, does not support the "FP--CQ complex hypothesis" for the antimalarial action of chloroquine.     

Evidence for electrogenic accumulation of mefloquine by malarial parasites

The uptake of mefloquine and chloroquine by Plasmodium chabaudi-infected mouse erythrocytes was measured in the presence and absence of ionophores and uncoupler in order to distinguish between the pH-dependent and pH-independent absorption of these drugs. Nigericin and CCCP (carbonylcyanide m-chlorophenylhydrazone) were used to relax the proton gradients and electrical potentials across the membranes. It was found that 40-60% of the mefloquine uptake, and 90% of the chloroquine uptake, was pH-dependent, the remainder being due to passive binding to cellular constituents. The distribution ratio of the pH-dependent uptake for mefloquine was about three times greater than for chloroquine. According to the lysosomotropic weak base hypothesis in which the neutral forms of weak bases are assumed to equilibrate across membranes, the mefloquine distribution should be smaller than the chloroquine distribution: since mefloquine is singly charged and chloroquine is doubly charged, the chloroquine distribution ratio should vary as the square of the mefloquine ratio. We interpret the greater uptake ratio of mefloquine to be evidence for the involvement of secondary active transport, with drug uptake being coupled to proton outflow by an antiporter protein. It is proposed that the uptake of mefloquine is electrogenic, with the proton gradient and the electrical potential both contributing to the driving force, but that the proton gradient alone is responsible for the chloroquine uptake.     

Oxygenated chalcones and bischalcones as a new class of inhibitors of DNA topoisomerase II of malarial parasites

The DNA topoisomerase (topo) II of chloroquine-sensitive and chloroquine-resistant strains of the rodent malaria parasite P. berghei were utilized as a target for testing of antimalarial compounds. Compounds belonging to the bischalcone and chalcone series significantly inhibited enzyme activity and percentage parasitaemia of chloroquine-sensitive and chloroquine-resistant strains of P. berghei. Compounds 1a, 1b, 2a, 2b, and 2c showed 100% inhibition while compounds 2h and 2i showed 60% and 63% inhibition of topoisomerase II activity of the chloroquine-sensitive strain, respectively. Compounds 2a, 2b, and 2d significantly inhibited the topo II activity of chloroquine-resistant strain. Compounds 2g and 2e specifically inhibited the topo II activity of the chloroquine-resistant strain of P. berghei with no effect on the chloroquine-sensitive strain. The in vitro topo II inhibition by chalcone and bischalcone analogs can be correlated with their in vivo antimalarial activity, as compounds 2c and 2h inhibited both in vitro activity of topo II and in vivo parasitaemia of the chloroquine-sensitive strain of P. berghei. In the chloroquine-resistant strain, compounds 2c, 2e, 2g, and 2i inhibited activity against both in vitro topo II and parasitaemia in vivo. The significant inhibition of topo II in the chloroquine-resistant strain by some of the analogs suggests the utilization of these structures for the synthesis of compounds active against chloroquine-resistant malarial parasites.     

Phosphono Analogues of Glutathione as New Inhibitors of Glutathione S-Transferases

Phosphono-analogues of glutathione containing the O=P(OR)₂ moiety in place of the cysteinyl residue CH₂SH 1a–1d were prepared by solution phase peptide synthesis. Benzyl, benzyloxycarbonyl, and tert-butyl protecting groups were used to mask the individual amino acid functional groups. The formation of peptide bonds was achieved by the usual peptide synthesis via activation of carboxylic functions with cyclohexylcarbodiimide and subsequent reaction with free amino groups. The thus obtained, fully-protected peptides were each purified by normal phase column chromatography. Deprotection was accomplished by hydrogenolysis and by treatment with HBr/acetic acid yielding the desired phosphonic acid diester 1a–1d . The inhibition of the glutathione conjugation of 1-chloro-2,4-dinitrobenzene by human placental glutathione S-transferase was studied by determining the IC₅₀ values of the new glutathione analogues. The IC₅₀ values were 291 μM, 139 μM, 64 μM, and 21 μM for the dimethyl, diethyl, diisopropyl, and di-n-butyl esters, respectively. The results clearly show that the formal substitution of the glutathione thiol function by phosphonic acid esters leads to a new class of glutathione S-transferase inhibitors. Further investigations directed at the question of whether or not these glutathione analogues are suitable for a modulation in chemotherapy are in progress.     

The hemolytic activity of citral--II. Glutathione depletion in citral treated erythrocytes

The role of glutathione in citral induced hemolysis was investigated. The concentration of intracellular glutathione diminished in citral treated erythrocytes. GSH disappeared before hemolysis started. Glucose inhibited citral induced hemolysis and restored the depleted cellular stores of GSH. The results presented confirm the previous suggestion that a free radical-peroxide mechanism is involved in citral induced hemolysis.     

Molecules of parasites as immunomodulatory drugs

Parasite molecules offer unique advantages for the treatment of immunologicical disorders, and several candidate molecules have been shown to be effective. In our studies, it was shown that a factor inducing immunoglobulin E from filarial nematode parasites was suppressive in animal models of immunological disorders such as allergy and insulin dependent diabetes mellitus (IDDM). The Th1/Th2 paradigm of CD4+ T helper cell subsets can provide the basis for the development of new types of drugs and of novel strategies for the treatment of allergic and autoimmune disorders by parasite molecules. In our experimental system, parasite molecules from a filarial nematode parasite led to the down-regulation of the allergic reaction in animal models. In the majority of hosts, infection with helminths is associated with markedly reduced cellular immune reactions and polarization of T cell responses to Th2 and Th3 types. Some studies have suggested that the stimulation of host immunoregulatory networks with parasite molecules leading to the synthesis of anti-inflammatory cytokines (interleukin10, transforming growth factor-beta (TGF-beta and others) can provide new therapy for immunological disorders. It is known that parasites produce some types of molecule that mimic host molecules such as CD40 ligand, TGF-beta and macrophage migration inhibitory factor. These molecules are also candidates for medicinal agents. This review describes many of the latest possibilities in this field and shows how they can be best put to use for the development of medicinal agents, molecular target identification, and for prioritization.     

Production of human liver prolidase by Saccharomyces cerevisiae as host cells

INTRODUCTION Mazur[1]first described the hydrolysis and detoxi-fication of diisopropylfluorophosphate (DFP) usingcrude preparations from human and rabbit tissues in1946. Organophosphoric acid anhydrolases (OPAA,EC 3.1.8.2) have been found in a wide variety ofprokaryotes and eukaryotes such as bacteria, protozoa,Altermonas haloplanktis[7] showed prolidase activity, Isolation of prolidase gene The human liversuggesting that the OPAA may be a prolidase[8]. These …     

Glutathione S-transferases as emerging therapeutic targets

Glutathione S-transferases (GST) represent a large family of Phase II detoxification enzymes widely expressed in animals and plants. These enzymes catalyse the conjugation of glutathione with some endogenous molecules and a broad range of exogenous substrates including various anticancer drugs. Due to high expression of GSTs in tumours when compared to normal tissues and their high level in plasma from cancer patients, these enzymes are considered to be cancer markers. Their involvement in resistance to anticancer drugs and an inverse correlation between expression and prognosis in many tumours provided a rationale for the design of inhibitors and prodrugs to enhance therapeutic index. The first generation of GST inhibitors included ethacrynic acid and showed promising potentiating activity in vitro but lack of isoenzyme specificity and diuretic side effects restricted clinical use. Novel GST inhibitors include glutathione analogues and demonstrate better specificities with fewer limiting toxicities. One lead compound is a potent inhibitor of the GSTP1-1 isoform in both cell lines and animal models. A GSTP1-1 activated prodrug has also been developed. Testing of the preclinical and clinical efficacy of these agents is presently in progress. Their rational design provides a promising new approach to targeting tumour-specific characteristics in a manner consistent with improving therapeutic index.     

Mast cells as critical effectors of host immune defense against Gram-negative bacteria

Mast cells are best known as central effector cells in IgE-mediated type I allergic diseases including asthma and hay fever. An increasing amount of evidence, however, has demonstrated that mast cells are sentinel cells playing a critical role in host defense against invading microbes. Mast cells are located immediately beneath the epithelial surfaces exposed to the outer environment, such as genitourinary and gastrointestinal tracts, skin, and airways. This review discusses recent studies on the critical roles of mast cells in host defense against Gram-negative bacterial infection. Mast cells are equipped with multiple receptors detecting the invading Gram-negative bacteria in both direct (opsonin-independent) and indirect (opsonin-dependent) mechanisms. The former includes Toll-like receptors (TLRs), CD48, and nucleotide-binding oligomerization (NOD) proteins, while the latter includes Fcγ receptors (FcγRs) and complement receptors. In addition to the detecting systems, mast cells are also armed with versatile tools to combat and kill Gram-negative bacteria. In response to the recognition of the Gram-negative bacterial infection, mast cells secrete various types of mediators which either regulate host immune system or directly attack the bacteria. Mast cells can also phagocytize and subsequently display the bacterial antigens on their cell surfaces. Moreover, recent findings have revealed the formation of extra-cellular traps by mast cells. Finally this review will especially focus on recent findings on LPS signaling in mast cells, both the functional outcome and the molecular mechanisms.     

Glutathione,glutathione-dependent enzymes and antioxidant status in erythrocytes from children treated with high-dose paracetamol

AIM: To investigate glutathione and antioxidant status changes in erythrocytes from febrile children receiving repeated supratherapeutic paracetamol doses. METHODS: Fifty-one children aged 2 months to 10 years participated in the study. Three groups were studied: group 1 (n = 24) included afebrile children who did not receive paracetamol; and groups 2 (n = 13) and 3 (n = 14) included children who had fever above 38.5 degrees C for more than 72 h. Patients in group 2 received paracetamol at a dose of 50 +/- 15 (30-75) mg kg(-1) day(-1) and those in group 3 received paracetamol above the recommended therapeutic dose, ie 107 28 (80-180) mg kg(-1) day(-1). A blood sample was taken for the measurement of liver transaminases, gammaglutamil transferase (GGT), reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST), superoxide dismutase (SOD) and antioxidant status. RESULTS: Aspartate aminotransferase activity in group 3 was higher than in the other groups (P = 0.027). GSH, SOD and antioxidant status were significantly lower in group 3 compared with groups 1 and 2 (mean differences: for GSH 3.41 micromol gHb(-1), 95% confidence interval (CI) 2.10-4.72, and 2.15 micromol gHb(-1), 95% CI 0.65-3.65, respectively; for SOD 856 U min(-1) gHb(-1), 95% CI 397-1316, and 556 U min(-1) gHb(-1), 95% CI 30-1082, respectively; and for antioxidant status 0.83 mmol l(-1) plasma, 95% CI 0.30-1.36, and 0.63 mmol l(-1) plasma, 95% CI 0.02-1.24, respectively). GR activity was significantly lower in groups 3 and 2 in comparison with group 1 (mean differences 3.44 U min(-1) gHb(-1), 95% CI 0.63-6.25, and 5.64 U min(-1) gHb(-1), 95% CI 2.90-8.38, respectively). Using multiple regression analysis, paracetamol dose was found to be the only independent variable affecting GR, GST and SOD activities (P = 0.007, 0.003 and 0.008, respectively). CONCLUSIONS: In febrile children, treatment with repeated supratherapeutic doses of paracetamol is associated with reduced antioxidant status and erythrocyte glutathione concentrations. These significant changes may indicate an increased risk for hepatotoxicity and liver damage.     

Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells.

Nordihydroguaiaretic acid (NDGA) induces apoptosis in a variety of cell lines. The mechanism(s) of this effect is not known, although the focus has been on the ability of NDGA to inhibit lipoxygenase (LOX) activities. In the present study, NDGA-induced apoptosis was studied in a murine hematopoietic cell line, FL5.12. Although this cell line lacks detectable LOX protein or activities, NDGA (10 microM) was able to induce apoptosis. There was a massive loss of mitochondrial membrane potential by 4 h after the addition of NDGA, suggesting that this organelle might be targeted by NDGA. A pro-oxidant NDGA effect has been suggested as playing a role in apoptosis. This was supported by the findings that glutathione disulfide levels were increased by 4 h following treatment with 10 microM NDGA, that pretreatment with N-acetylcysteine completely blocked the NDGA-induced loss of membrane potential and apoptosis, and that lipid peroxidation was enhanced in cells treated with NDGA. However, no evidence of increased levels of reactive oxygen could be seen in NDGA-treated cells loaded with dichlorofluorescin diacetate or dihydrorhodamine and analyzed by flow cytometry. Bcl-X(L) protein levels were unaffected by NDGA treatment. Caspase-3 was rapidly activated with a peak at 8 h after FL5.12 cells were treated with NDGA. Ac-DEVD-CHO (25 microM) and boc-asp-FMK (20 microM) both inhibited caspase-3 enzyme activity by 97% 8 h after NDGA treatment. Boc-asp-FMK, a more general caspase inhibitor, delayed NDGA-induced apoptosis while Ac-DEVD-CHO, a more specific inhibitor of caspase-3, had no effect. These results suggest that NDGA-induced apoptosis happens through reactions that depolarize mitochondria, oxidize glutathione and lipids, but do not generate significant amounts of free reactive oxygen species.     

Digestion of the host erythrocyte by malaria parasites is the primary target for quinoline-containing antimalarials

Intraerythrocytic malaria parasites feed on their host cell cytosol. We show that human red blood cells infected with the malaria parasite Plasmodium falciparum, produce free amino acids the composition of which resembles that of globin, the most abundant red blood cell protein. The rate of amino acid production is almost equal to the rate of efflux of these acids from the infected cell. Production of amino acids increases with parasite age: the rates of production at the young ring and the mature trophozoite stages were 3.3 and 13.5 nmol/10(8) infected cells per min at 37 degrees, respectively, compared with 0.04 nmol/10(8) cells per min in uninfected cells. The quinoline-containing antimalarial drugs, chloroquine, quinine and mefloquine, inhibit amino acid production at the same concentrations at which they inhibit parasite growth, but have no effect on the endogenous parasite protein degradation. We suggest that parasite feeding on host cell cytosol is the primary target for the antimalarial action of these drugs. Chloroquine accumulation, the rate of amino acid production by infected cells and the inhibitory effect of the drug, were determined simultaneously at the different stages of parasite development. At all stages the rate of amino acid production and chloroquine accumulation were directly related and both were inversely related to the inhibitory efficiency of the drug. The lysosomotropic agents methylamine and NH4Cl at millimolar concentrations also inhibit amino acid production, suggesting that the process is pH dependent and localized in the vacuole. Host cytosol degradation and drug accumulation both take place in the parasite food vacuole. Our observations imply that the metabolically dependent acidification of this parasite organelle is involved in both processes.     

Glutathione transferases in hepatocyte-like cells derived from human embryonic stem cells.

Human embryonic stem cells (hESCs) offer a potential unlimited source for functional human hepatocytes, since hESCs can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing several hepatic markers. These hepatocyte-like cells could be used in various human in vitro hepatocyte assays, e.g. as a test system for studying drug metabolism and drug-induced hepatotoxicity. Since the toxic effect of a compound is commonly dependent on biotransformation into metabolites, the presence of drug metabolising enzymes in potential test systems must be evaluated. We have investigated the presence of glutathione transferases (GSTs) in hepatocyte-like cells by immunocytochemistry and Western blotting. Results show that these cells have high levels of GSTA1-1, whereas GSTP1-1 is not present in most cases. GSTM1-1 is detected by immunocytochemistry but not by Western blotting. In addition, GST activity is detected in hepatocyte-like cells at levels comparable to human hepatocytes. These results indicate that the hepatocyte-like cells have characteristics that closely resemble those of human adult hepatocytes.     

The plastid-like organelle of apicomplexan parasites as drug target

Apicomplexan parasites infectious to humans include Plasmodium spp., Babesia spp., Toxoplasma gondii, Cryptosporidium spp., Isospora belli and Cyclospora cayetanensis. With exception of Cryptosporidium spp., these parasites possess a non-photosynthetic plastid-like organelle called apicoplast. The apicoplast possesses a small circular genome and harbours prokaryotic-type biochemical pathways. As the most important metabolic functions, the mevalonate independent 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid synthesis and the type II fatty acid synthesis system are operative inside the apicoplast. Classical antibacterial drugs such as ciprofloxacin, tetracycline, doxycycline, clindamycin and spiramycin inhibit the apicoplast-located gyrase and translation machinery, respectively, and are currently used in the clinic for the treatment of infections with apicomplexan parasites. As an inhibitor of isoprenoid synthesis, fosmidomycin was proven to be effective against acute P. falciparum malaria in clinical phase II studies. Triclosan, an inhibitor of fatty acid synthesis, was active in a malaria mouse model. In vitro antimalarial activity was shown for inhibitors of peptide deformylase and the import of apicoplast-targeted proteins. Work on various other inhibitors of apicoplast-located biochemical processes is ongoing.     

Glutathione consumption and inactivation of glutathione-related enzymes in liver, erythrocytes and serum of rats after methanol intoxication

The primary metabolic fate of methanol is oxidation to formaldehyde and then to formate. These processes are accompanied by formation of superoxide anion and further hydrogen peroxide. Glutathione plays a unique role in the cellular defence system against xenobiotics. The glutathione (GSH) content and glutathione peroxidase (GSH-Px) and glutathione reductase (GSSG-R) activities were measured in liver, erythrocytes and serum of rats. Rats were intoxicated with 3.0 and 6.0 g methanol/kg body wt. and measurements taken after 6, 12 and 24 h and 2, 5 and 7 days of intoxication. The decrease in GSH content and in GSH-related enzyme activity was observed during the whole time-course of the intoxication. The most significant changes were observed in the erythrocytes. The results obtained show that the protection against oxidative damage due to methanol intoxication in rats seems to be less efficient than in control rats. Received: 7 January 1997 / Accepted: 26 May 1997     

Cationic nanoemulsion as a delivery system for oligonucleotides targeting malarial topoisomerase II

A promising strategy based on the antisense oligonucleotides against the Plasmodium falciparum topoisomerase II has been considered using cationic nanoemulsion as oligonucleotide delivery system. Phosphodiester and chemically modified phosphorothioate oligonucleotides bearing negative charges were adsorbed on positively charged emulsion composed of medium chain triglycerides, egg lecithin, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and water, at different +/− charge ratios (positive charges from cationic lipid/negative charges from oligonucleotide): +0.5/−, +2/−, +4/− and +6/−. The physicochemical properties of the complexes were determined, as well as their stability in culture medium. Their interaction with erythrocytes through hemolysis, binding experiments and confocal microscopy were also evaluated. Finally, the in vitro evaluation of parasite growth and reinfection capacity was performed. The overall results showed that antisense oligonucleotides against P. falciparum topoisomerase II gene can be efficiently adsorbed onto a cationic nanoemulsion forming complexes. Whereas unloaded nanoemulsion displayed an hemolytic effect due to the presence of the cationic lipid, this was not the case of loaded nanoemulsion at low +/− ratios. Oligonucleotide-loaded nanoemulsions were found to be located inside the infected erythrocytes, inhibiting efficiently parasite growth (until 80%) and causing a delay in P. falciparum life cycle.