Expression and characterization of the ATP-binding domain of a malarial Plasmodium vivax gene homologous to the B-subunit of the bacterial topoisomerase DNA gyrase

We have previously reported the presence of a DNA gyrase-like topoisomerase activity associated with the 35kb apicoplast DNA in the malarial parasite Plasmodium falciparum [Weissig V, Vetro-Widenhouse TS, Rowe TC. Topoisomerase II inhibitors induce cleavage of nuclear and 35kb plastid DNAs in the malarial parasite Plasmodium falciparum. DNA Cell Biol 1997;16:1483]. Sequences encoding polypeptides homologous to both the A and B subunits of bacterial DNA gyrase have been identified in the genome sequence of P. falciparum among data produced by the Malaria Genome Consortium and the University of Florida Malaria Gene Sequence Tag Project. Based on these findings, we have cloned and expressed a region of the Plasmodium vivax GyrB gene encoding a 43kDa polypeptide homologous to the ATP-binding domain of Escherichia coli DNA gyrase. The 43kDa PvGyrB polypeptide was found to have intrinsic ATPase activity with a K(m) of 0.27mM and a k(cat) of 0.051s(-1). The PvGyrB ATPase was also sensitive to the bacterial DNA gyrase inhibitor coumermycin. The implications of these findings are discussed.     

Clinical application of fast protein liquid chromatography (FPLC) system

FPLC system is a high-performance liquid chromatography system, designed specifically for separation of proteins, peptides and polynucleotides. Many kinds of column have been prepared, including Mono Q, Mono S, Mono P, Superose, etc. for ion exchange chromatography, chromatofocussing, gel filtration, hydrophobic chromatography, reversed phase chromatography and affinity chromatography. It is a fast and simple system for separation of proteins in various body fluids, particularly plasma, urine and cerebrospinal fluid.     

Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum

A major obstacle in the global effort to control malaria is the paucity of anti-malarial drugs. This is compounded by the continuing emergence and spread of resistance to old and new anti-malarial drugs in the malarial parasites. Here we describe the anti-malarial effect of phosphorothioate antisense (AS) oligodeoxynucleotides (ODNs) targeting the aldolase enzyme of Plasmodium falciparum, using the asexual blood stages of the parasite grown in vitro. The blood stages of P. falciparum depend almost entirely on the energy produced by their own glycolysis. Aldolase, the fourth enzyme of the glycolytic pathway, is highly upregulated during the malarial 48-h life cycle. We found that the mRNA of this enzyme can be inhibited, in a sequence specific manner, using AS-ODN to the splice sites on the pre-mRNA of malarial aldolase. At the enzyme level, both specific AS-ODNs for the splice sites, as well as for the translation initiation site on mature mRNA, can inhibit aldolase enzyme activity within the trophozoites of P. falciparum. Furthermore, this downregulation of the malarial aldolase results in a reduction in the production of ATP within the parasite. Finally, the treatment reduces parasitemia. In summary, AS-ODNs targeting the aldolase gene of P. falciparum can interfere with the blood-stage life cycle of this parasite in vitro by inhibiting the expression of the enzyme aldolase which results in decreased malarial glycolysis and energy production. Thus, we conclude that blockade of the expression of malarial glycolytic enzymes using specific AS-ODNs has the potential of a new anti-malarial strategy.     

Induction of nucleoside transport sites into the host cell membrane of Babesia bovis infected erythrocytes

Normal bovine erythrocytes have negligible ability to transport adenosine and related nucleosides across their cell membrane. However, infection with the intraerythrocytic parasite Babesia bovis was found to induce a nucleoside permeation site into the host cell membrane. Transport experiments over periods of up to 30 s determined that the transport rate of 1 microM adenosine into the infected cell was 1.72 +/- 1.2 pmol incorporated (microliter cell water)-1s-1, a rate three times higher than for normal human erythrocytes. Incorporation studies over 6 h with labelled adenosine indicated that the purine moiety was incorporated into parasite nucleic acids. The mammalian nucleoside transport inhibitors, nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole inhibited the induced nucleoside transport mechanism in the Babesia-infected erythrocytes, though at higher concentrations than those required to inhibit normal human erythrocyte transport. An ID50 value for NBMPR of 0.36 microM was determined. Phloretin and 5'-p-fluorosulphonyl benzoyl adenosine-HCl (5FSBA) were also shown to be inhibitory, with ID50 values of 0.11 and 0.18 microM, respectively, whilst phlorizin and verapamil at 1 microM had no effect. Binding studies with [3H]NBMPR indicated that high-affinity NBMPR binding sites could not be detected in either normal or B. bovis infected bovine erythrocytes. The results indicate that the induced nucleoside permeation site(s) in B. bovis infected erythrocytes has characteristics different from either human erythrocytes or erythrocytes infected with the malarial parasites Plasmodium falciparum or Plasmodium yoelii.     

Purification of hypoxanthine-guanine phosphoribosyltransferase of Plasmodium lophurae

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was isolated from the malarial parasite, Plasmodium lophurae. The apparent pI, as determined by chromatofocusing, was 7.6. The native molecular weight was 79,000. The pH profile of HGPRT exhibited a broad pH optimum. With hypoxanthine as substrate maximal activity was achieved from pH 6.0-10.0, and with guanine as substrate maximal activity occurred from pH 7.5-9.5. The enzyme exhibited Michaelis-Menten kinetics with all substrates. The Km values were 3.8 microM (hypoxanthine), 2.4 microM (guanine), 6.2 microM (6-mercaptopurine), 7.6 microM (6-thioguanine), and 360 microM (8-azahypoxanthine). 6-Thioinosine, 9-beta-arabinofuranosylhypoxanthine, 6-chloropurine, xanthine and azaguanine were inhibitors of the P. lophurae enzyme. From the substrate and inhibitor data it appears that the sixth position on the purine ring plays a major role in enzyme activity.     

Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species

GTP cyclohydrolase (EC 3.5.4.16), the first enzyme in the pteridine pathway leading to the de novo formation of folic acid, has been identified and isolated from the human malaria parasite, Plasmodium falciparum. The enzyme was purified 200-fold by high performance size-exclusion chromatography on a TSK-G-3000 SW protein column. The molecular weight was estimated at 300 000. Optimal enzyme activity was observed at pH 8.0 and 42 degrees C. The Km for GTP was 54.6 microM. Products of the enzyme reaction were identified as the carbon-8 of GTP and D-erythro-dihydroneopterin triphosphate. ATP was a competitive inhibitor (Ki = 600 microM) of the enzyme. Activity of the enzyme was Mg2+-independent, whereas Mn2+, Cu2+ and Hg2+ (5 mM) were inhibitory. GTP cyclohydrolase activity was also identified in a murine parasite, Plasmodium berghei, and a simian parasite, Plasmodium knowlesi. Activity of the enzyme in P. knowlesi, an intrinsically synchronous quotidian parasite, was found to be dependent on the stage of parasite development.     

Glycoproteins present in human follicular fluid that inhibit the zona- binding capacity of spermatozoa

Previous studies have suggested that human follicular fluid contains factors that reduce the zona-binding capacity of spermatozoa. The present study provides further evidence of the existence of such factors. Using the hemizona binding assay (HZA), we have shown that the inhibitory effect of human follicular fluid on the zona-binding capacity of spermatozoa is concentration-dependent, an inhibitory effect being detected when the concentration of human follicular fluid was > or = 10%. A 1% concentration of human follicular fluid did not possess this inhibitory activity. Heating human follicular fluid at 56 degrees C for 30 min did not affect its inhibitory properties; treatment with proteinase-K abolished such inhibition. Human follicular fluid was fractionated sequentially by concanavalin-A affinity chromatography, Mono Q ion-exchange chromatography and Superose-12 gel filtration. The zona binding inhibitory activity resided in the fraction which bound to the lectin and Mono Q column and contained molecules with native molecular weights of 32 and 192 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis suggested that the 192 kDa glycoprotein was a tetramer, while the 32 kDa glycoprotein remained as a single molecular species under denaturing conditions. We conclude that two glycoproteins were responsible for the zona binding inhibitory activity of human follicular fluid. The physiological role of these factors remains unclear.      

In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo

The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID50 values (concentration of compound causing 50% inhibition of [3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 +/- 8.7, 6.4 +/- 5.3 and 6.3 +/- 0.5 microM, respectively. Comparative ID50 values for chloroquine were 13.5 +/- 4.2, 22.8 +/- 7.6 and 343 +/- 114 microM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 microM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. 2.4.2.10) and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5'-phosphate decarboxylase (E.C. 4.1.1.23).     

Molecular cloning and characterization of Plasmodium falciparum transketolase

The pentose phosphate pathway (PPP) is an important metabolic pathway for yielding reducing power in the form of NADPH and production of pentose sugar needed for nucleic acid synthesis. Transketolase, the key enzyme of non-oxidative arm of PPP, plays a vital role in the survival/replication of the malarial parasite. This enzyme in Plasmodium falciparum is a novel drug target as it has least homology with the human host. In the present study, the P. falciparum transketolase (PfTk) was expressed, localized and biochemically characterized. The recombinant PfTk harboring transketolase activity catalyzed the oxidation of donor substrates, fructose-6-phosphate (F6P) and hydroxypyruvate (HP), with K(m)(app) values of 2.25 and 4.78 mM, respectively. p-Hydroxyphenylpyruvate (HPP) was a potent inhibitor of PfTk, when hydroxypyruvate was used as a substrate, exhibiting a K(i) value of 305 microM. At the same time, noncompetitive inhibition was observed with F6P. The native PfTk is a hexamer with subunit molecular weight of 70kDa, which on treatment with low concentrations of guanidine hydrochloride (GdmCl) dissociated into functionally active dimers. This protein was localized in the cytosol and nucleus of the parasite as studied by confocal microscopy. A model structure of PfTk was constructed based on the crystal structure of the transketolases of Saccharomyces cerevisae, Leishmania mexicana and Escherichia coli to assess the structural homology. Consistent with the homology modeling predictions, CD analysis indicated that PfTk is composed of 39% alpha-helices and 26% beta-sheets. The availability of a structural model of PfTk and the observed differences in its kinetic properties compared to the host enzyme may facilitate designing of novel inhibitors of PfTk with potential anti-malarial activity.     

The trypanocidal Cape buffalo serum protein is xanthine oxidase.

Plasma and serum from Cape buffalo (Syncerus caffer) kill bloodstream stages of all species of African trypanosomes in vitro. The trypanocidal serum component was isolated by sequential chromatography on hydroxylapatite, protein A-G, Mono Q, and Superose 12. The purified trypanocidal protein had a molecular mass of 150 kDa, and activity correlated with the presence of a 146-kDa polypeptide detected upon reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences of three peptide fragments of the 146-kDa reduced polypeptide, ligand affinity and immunoaffinity chromatography of the native protein, and sensitivity to pharmacological inhibitors, identified the trypanocidal material as xanthine oxidase (EC 1.1.3.22). Trypanocidal activity resulted in the inhibition of trypanosome glycolysis and was due to H2O2 produced during catabolism of extracellular xanthine and hypoxanthine by the purine catabolic enzyme.     

Myristoylated adenylate kinase-2 of Plasmodium falciparum forms a heterodimer with myristoyltransferase

Adenylate kinases (AK; ATP+AMP<-->2 ADP; E.C. 2.7.4.3.) are enzymes essentially involved in energy metabolism and macromolecular biosynthesis. As we reported previously, the malarial parasite Plasmodium falciparum possesses one genuine AK and one GTP-AMP phosphotransferase. Analysis of the P. falciparum genome suggested the presence of one additional adenylate kinase, which we designated AK2. Recombinantly produced AK2 was found to be a monomeric protein of 33 kDa showing a specific activity of 10 U/mg with ATP and AMP as a substrate pair and to interact with the AK-specific inhibitor P(1),P(5)-(diadenosine-5')-pentaphosphate (IC(50)=200 nM). At its N-terminus AK2 carries a predicted myristoylation sequence. This sequence is only present in AK2 of P. falciparum causing the severe tropical malaria and not in other malarial parasites. We heterologously coexpressed AK2 and P. falciparum N-myristoyltransferase (NMT) in the presence of myristate in Escherichia coli. As demonstrated by protein purification and mass spectrometry, AK2 is indeed myristoylated under catalysis of the parasites' transferase. The modification significantly enhances the stability of the kinase. Furthermore, AK2 and NMT were shown to interact strongly with each other forming a heterodimeric protein in vitro. To our knowledge this is the first direct evidence that P. falciparum NMT myristoylates an intact malarial protein.     

Phosphofructokinase from Dirofilaria immitis: effect of fructose 2,6-bisphosphate and AMP on the non-phosphorylated and phosphorylated forms of the enzyme

The enzyme responsible for the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), 6-phosphofructo-2-kinase, was shown to be present in the heart worm, Dirofilaria immitis. The level of Fru-2,6-P2 was determined to be 4 +/- 0.3 nmol(g wet weight)-1 in the tissues of the filariid. Fru-2,6-P2 stimulated the activity of both the non-phosphorylated and phosphorylated forms of the D. immitis phosphofructokinase (PFK). The Kact values for Fru-2,6-P2 were 378 +/- 18 nM and 65 +/- 6 nM for the non-phosphorylated and phosphorylated forms, respectively, at 1 mM fructose 6-phosphate (Fru-6-P) and 1 mM ATP at pH 6.8. AMP also stimulated the activity of both forms of the enzyme with Kact values of 230 +/- 10 microM and 37.3 +/- 6.1 microM for the non-phosphorylated and phosphorylated forms, respectively. In the absence of any effectors, the S0.5 values for Fru-6-P were 17.4 mM and 11.0 mM for the non-phosphorylated and phosphorylated forms, respectively, of the D. immitis PFK at 1 mM ATP, pH 6.8. These S0.5 values were lowered to 0.03 mM by the combined effects of saturating levels of Fru-2,6-P2 and AMP. A physiological assay was developed based on the level of metabolites in the parasite that influence the activity of PFK. This assay contained the known effectors of the PFK at concentrations approximating those found in the parasite. Under these conditions the KFru-6-P values were 153 microM and 60 microM for the non-phosphorylated and phosphorylated forms of the PFK, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of 37-kilodalton proteases from Plasmodium falciparum and Plasmodium berghei which cleave erythrocyte cytoskeletal components

Cytosoluble 100,000 X g extracts from Plasmodium berghei or Plasmodium falciparum infected red blood cells were shown to hydrolyze erythrocyte spectrin. By Fast Protein Liquid Chromatography (FPLC), these enzymes were purified and exhibited a pI of 4.5 and Mr of 37,000 using SDS-PAGE under reducing conditions. An immunochemical enzyme assay using anti-spectrin antibodies was developed. The optimal activity using spectrin as substrate was at pH 5.0, and the enzymes were strongly inhibited by HgCl2, ZnCl2, chymostatin, leupeptin and aprotinin, and moderately by pepstatin. These properties of the Pf37 and Pb37 proteases differ from the Plasmodium lophurae and P. falciparum 'cathepsin D-like' enzymes and from the serine or cysteine neutral proteases previously described in P. falciparum and P. berghei infected red blood cells. While the Pf37 and Pb37 enzymes cleaved spectrin preferentially, degradation of band 4.1 was also observed with high concentration of enzyme. The parasite origin of the Pf37 protease was clearly demonstrated, since purified radiolabeled enzyme was active on spectrin. A high-molecular-weight polymer (greater than 240 kDa) was often observed on incubating purified spectrin and Pf37 protease. The breakdown of erythrocyte cytoskeletal components could be of interest in the release of merozoites from segmented schizonts or during the process of invasion of erythrocytes by merozoites.     

Further characterization and partial amino acid sequence of a cysteine proteinase from Trypanosoma cruzi

A cysteine proteinase from epimastigotes of Trypanosoma cruzi, Tul 2 stock, has been purified to homogeneity from cell-free extracts obtained by freezing and thawing, by a procedure involving ammonium sulfate fractionation, DEAE-Sephacel chromatography, and gel filtration on Sephadex G-200; when necessary, further purification was attained by fast protein liquid chromatography on Mono Q and Superose 6 columns. The purified enzyme was strongly inhibited by leupeptin, antipain and chymostatin (I50 values of 0.25, 0.75 and 1 microM, respectively), little inhibited by elastatinal, and unaffected by pepstatin A. The enzyme is a glycoprotein, as shown by binding to ConA-Sepharose and elution with alpha-methyl-D-mannopyranoside and alpha-methyl-D-glucopyranoside. Partial amino acid sequences were obtained from the N-terminal end (32 amino acids) of the carbamidomethylated enzyme, and from a tryptic peptide (14 amino acids) of the pyridylethylated enzyme. Both regions show considerable homology with papain and some cathepsins, such as cathepsin L, thus showing that the enzyme belongs to the cysteine proteinase family.     

Purification and characterization of cell wall proteins of Listeria monocytogenes

Two proteins were purified from Listeria monocytogenes cell wall using detergent extraction, Superose 6 gel chromatography. Mono Q cation-exchange chromatography and Superose 12 gel chromatography. Proteins were shown to form complexes of ca. 300 kDa and pI 4.7. These complexes could not be dissociated in 6 M urea, however, during SDS-PAGE 79 and 39 kDa monomers were formed. Immunoblot analysis showed that the proteins under investigation were common to all listerial strains tested, but were absent in strains of other bacteria. We propose that the proteins investigated here could serve as immunoserological markers for Listeria.     

Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

The properties of a purine phosphoribosyltransferase from late trophozoites of the human malaria parasite, Plasmodium falciparum, are described. Enzyme activity with hypoxanthine, guanine and xanthine as substrates eluted in parallel during hydroxylapatite, size exclusion and DEAE-Sephadex chromatography as well as during chromatofocusing experiments. Furthermore, enzyme activity with all three purine substrates changed in parallel during heat inactivation of enzyme preparations and upon cold storage (4 degrees C) of the enzyme. When considered together, these results support the view that the phosphoribosyltransferase is capable of utilizing all three purine bases as substrates. Additional characterization revealed that the apparent molecular weight and isoelectric point of this enzyme are 55,500 and 6.2, respectively, and that the apparent Km for 5-phosphoribosyl-1-pyrophosphate ranges from 13.3 to 21.4 microM, depending on the purine base serving as substrate. The apparent Km values for hypoxanthine, guanine and xanthine were found to be 0.46, 0.30 and 29 microM, respectively. Other experiments showed that several divalent cations and sulfhydryl reagents produce a marked reduction of enzyme activity whereas dithiothreitol activates the enzyme. It should be noted that the ability to utilize xanthine as a substrate serves to distinguish the P. falciparum enzyme from its counterpart in the parasite's host cell, the human erythrocyte. The human enzyme shows only barely detectable activity with xanthine while the parasite enzyme displays similarly high levels of activity with all three purine substrates. Thus, the parasite enzyme might prove to be selectively susceptible to inhibition by xanthine analogs and related compounds.     

Purification of monoclonal antibodies raised against prostate-specific acid phosphatase for use in vivo in radioimaging of prostatic cancer

In developing diagnostic reagents for the radioimaging of prostatic cancer, methods were optimized for the purification of two mouse IgG1 monoclonal antibodies raised against prostate-specific acid phosphatase and produced in cell culture. Two different two-step methods were selected. One method consisted of two successive ion exchange chromatographic steps on Mono S and Mono Q; in the other method, Mono S chromatography was followed by hydrophobic interaction (Alkyl Superose) chromatography. In both cases, fast protein liquid chromatography (FPLC) instrumentation was used. The antibodies were purified from cell culture media containing fetal calf serum (1-5%). Highly pure (greater than 95%) IgG1 antibodies, free of contaminating serum-derived proteins or column materials, were obtained in good yield (greater than 90% recovery). The purified antibodies completely retained their immunological reactivity towards prostate-specific acid phosphatase and were sterile and pyrogen-free. Since the monoclonal antibodies produced were intended for applications in vivo, an essential feature of the methods selected was the availability of in situ cleaning procedures for sterilization of the gel materials and for the inactivation of viruses and pyrogens in the gels. The methods developed could be readily scaled up for preparative purposes.     

Cytochrome P-450 activity in malarial parasites and its possible relationship to chloroquine resistance

Cytochrome P-450-dependent enzyme activities have been determined in malarial parasites. Both Plasmodium berghei and Plasmodium falciparum parasites exhibited activity and these activities were greater in chloroquine resistant parasites than in sensitive strains. This enzyme activity could be induced by phenobarbitone and inhibited by specific inhibitors of the cytochrome P-450 family of enzymes. The significance of these observations in parasite drug resistance is discussed.     

Coenzyme Q10 in dilated cardiomyopathy

The authors have tried to study the therapeutic efficacy of coenzyme Q10 (CoQ10) in patients with dilated cardiomyopathy (DCM). In fact, CoQ10 has been shown to be deficient in myocardial tissue biopsies taken from DCM hearts, compared to normal hearts. Thirty patients with histological diagnosis of DCM were orally treated with CoQ10 (100 mg/die) for 2 months. Before and after treatment a clinical examination with determination of NYHA class and an echocardiographic examination with determination of ejection fraction (EF) and of telediastolic (TDV) and telesystolic (TSV) volumes were performed, and blood was drawn for plasma CoQ10 determination. In seven patients the pretreatment endomyocardial level of CoQ10 was also assayed. Seven patients left the study because of poor therapeutic compliance. In 47% of patients the clinical symptomatology regressed, with improvement of NYHA class. The EF improved from 0.31 +/- 0.09 to 0.37 +/- 0.11 (p less than 0.001). The TDV passed from 262.2 +/- 85 ml to 203.3 +/- 83 ml (p less than 0.05), and the TSV from 166.13 +/- 75 ml to 126.9 +/- 56 ml (ns). The CoQ10 plasmatic levels improved in 95% of the patients: from 0.74 +/- 0.37 micrograms/ml to 2.27 +/- 0.99 micrograms/ml (p +/- 0.0001). The CoQ10 myocardial levels did not show univocal values, but the patients with lower myocardial levels seemed to have a better therapeutic response. These data suggest that the CoQ10 deficiency in DCM may be reversible and that the therapeutic effects depend on the basal plasmatic and myocardial levels. Therapy with coenzyme Q10 may be considered to be an efficacious aid in the traditional treatment of chronic cardiac failure.