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.     

Identification, expression, localization and serological characterization of a tryptophan-rich antigen from the human malaria parasite Plasmodium vivax

Plasmodium vivax is most common but non-cultivable human malaria parasite which is poorly characterized at the molecular level. Here, we describe the identification and characterization of a P. vivax Tryptophan-Rich Antigen (PvTRAg) which contains unusually high (8.28%) tryptophan residues and is expressed by all blood stages of the parasite. The pvtrag gene comprises a 978bp open reading frame interrupted by two introns. The first intron is located in the 5'-untranslated region while the second one is positioned 174bp downstream to the ATG codon. The encoded approximately 40kDa protein contains a transmembrane domain near the N-terminus followed by a tryptophan-rich domain with significantly high surface probability and antigenic index. It is localized in the parasite cytoplasm as well as in the cytoplasm of the parasitized erythrocyte. The purified E. coli expressed recombinant PvTRAg protein showed a very high seropositivity rate for the presence of antibodies amongst the P. vivax patients, indicating that the antigen generates significant humoral immune response during the natural course of P. vivax infection. Analysis of various field isolates revealed that the tryptophan-rich domain is highly conserved except for three-point mutations. The PvTRAg could be a potential vaccine candidate since similar tryptophan-rich antigens of P. yoelii have shown protection against malaria in murine model.     

Human TRP14 gene homologue from amphioxus Branchiostoma belcheri: identification, evolution, expression and functional characterization

Thioredoxin-related protein of 14 kDa, TRP14, has previously been identified only in humans. Here we report the identification and expression of an amphioxus TRP14 gene, named AmphiTRP14, the first such data in a non-mammalian organism. AmphiTRP14 consists of a 372-bp open reading frame coding for a 123-amino-acid protein with a calculated molecular weight of 14 kDa. It shares 56% identity with human TRP14 and possesses a highly conserved motif CPDC. Sequence comparison suggests the evolutionary appearance of the four-exon-three-intron organization of TRP14 genes after the split of protostome/deuterostome, which is highly conserved since then. AmphiTRP14 has been successfully expressed in Escherichia coli and purified. The recombinant protein exhibited features characteristic of human TRP14, including a reductase activity towards insulin. Both in situ hybridization histochemistry and immunohistochemistry revealed that AmphiTRP14 was expressed in a tissue-specific manner, with the most abundant expression in the hepatic caecum, ovary and hind-gut. This suggests that AmphiTRP14 plays a fundamental but tissue-specific role, or alternatively reflects differences in the tissue susceptibility to oxidative damage.     

Isolation and characterization of 5-fluorouracil-resistant mutants of Chinese hamster ovary cells deficient in the activities of orotate phosphoribosyltransferase and orotidine 5′-monophosphate decarboxylase

A rapid, simple method for isolation of large numbers of Chinese hamster ovary cell (CHO-K1) mutants deficient in the final two enzymes of UMP biosynthesis, orotate phosphoribosyltransferase (EC 2.4.2. 10), and OMP decarboxylase (EC 4.1.1.23) is described. The method takes advantage of the fact that CHO-K1 cells require orotate phosphoribosyltransferase to activate the pyrimidine analog 5-fluorouracil to its active form; hence, mutants lacking this enzyme activity can be isolated simply by selection for cells resistant to 5-fluorouracil. The relevance of these observations to the metabolism of this cancer chemotheraputic agent, to the study of the structure of the protein(s) involved in catalyzing the last two steps of UMP biosynthesis, to the study of the structure of the gene(s) coding for this protein, and to analysis of the human genetic disease orotic aciduria is discussed.Number 5 in the series: Biochemical Analysis of Pyrimidine Biosynthesis in Mammalian Cells.     

History, dynamics, and public health importance of malaria parasite resistance

Despite considerable efforts, malaria is still one of the most devastating infectious diseases in the tropics. The rapid spread of antimalarial drug resistance currently compounds this grim picture. In this paper, we review the history of antimalarial drug resistance and the methods for monitoring it and assess the current magnitude and burden of parasite resistance to two commonly used drugs: chloroquine and sulfadoxine-pyrimethamine. Furthermore, we review the factors involved in the emergence and spread of drug resistance and highlight its public health importance. Finally, we discuss ways of dealing with such a problem by using combination therapy and suggest some of the research themes needing urgent answers.     

Human cerebral malaria: characterization of malarial antibodies in cerebrospinal fluid.

Anti-malarial antibodies were quantified in cerebrospinal fluid (CSF) of 17 cases of cerebral malaria, 16 presumptive cases (no demonstrable parasitaemia in peripheral blood but responding to i.v. quinine therapy) of cerebral malaria, and 15 controls. A schizont-enriched Plasmodium knowlesi antigen was used in an ELISA. Anti-malarial antibodies of IgA and IgM isotypes were not detectable in most of the CSF samples analysed, although serum antibody titres were high. However, 88% of CSF from cerebral malaria and 56% of presumptive cerebral malaria cases had significant levels of IgG anti-malarial antibodies in comparison to control CSF. The antibody levels did not correlate with the severity of coma but correlated well with the duration of coma. The CSF malarial antibody titres were independent of degree of parasitaemia. The possible role of CSF anti-malarial antibodies in cerebral malaria in the light of recent demonstrations of intrathecal synthesis of immunoglobulins and deposition of immune complex in cerebral tissues is discussed.     

Human peripheral Null lymphocytes I. Isolation,immunological and functional characterization

A Null lymphocyte-enriched population was isolated from human peripheral blood of healthy donors using a combination of Ficoll density gradient centrifugation followed by elimination of mononuclear phagocytes, passage through Ig-anti-Ig columns and sedimentation of E rosettes. After each separation step lymphocyte fractions were examined for morphology, cell surface markers, mitogen responsiveness and effector functions in antibody-dependent (ADCC) and spontaneous cellular cytotoxicity (SCMC) reactions against an allogeneic melanoma cell line. The final Null lymphocyte preparation was recovered at a rate of 1 to 3 % from the population passed through Ig-anti-Ig columns (fraction FFF-C). The marker analysis revealed over 99 %of surface Ig-negative lymphoid cells; 50 to 60 % of these cells were ‘real Null’ cells lacking immunological cell surface markers, 7 % formed EA, 13 % EAC and 24 %E rosettes. Regarding the mitogen responses, passage through Ig-anti-Ig columns drastically reduced concanavalin A (Con A), pokeweed mitogen (PWM)and tuberculin (PPD) responses, whereas the phytohemagglutinin (PHA) response was reduced in absolute counts but not in the stimulation index. Compared to the T cell-enriched lymphocyte fraction, the Null cells showed significantly diminished proliferative responses to PHA and Con A and slightly increased reactivity to PWM and PPD. Although depleted of high affinity Fc receptor lymphocytes, the Null cell fractions exhibited good ADCC and SCMC activities being about 4 to 6 times higher than in the T cell fraction.     

Human peripheral blood accessory cell: isolation by hypotonic density gradient, functional, and phenotypical characterization

In order to purify the human peripheral blood-derived accessory cell that cooperate with T lymphocytes in the process of mitogenic stimulation, we developed a new density gradient separation. This was based on the principle of hypotonic swelling of the cells to obtain a differential change of the buoyant densities of cells. By this method, we have obtained a highly accessory cell-depleted lymphocyte fraction whose proliferative response to sodium periodate stimulation was almost aborted. Another fraction containing high accessory cell activity was further divided into Fc-receptor-positive and -negative cells. The latter revealed the highest accessory activity for T lymphocyte periodate stimulation. The cells were characterized according to a number of markers and appeared to resemble lymphoid dendritic cells. Compared with the monocyte/macrophage fraction, they showed veils and dendritiform elongations and expressed reduced values of monocyte/macrophage specific markers. Compared with the high accessory activity of these cells, monocytes/macrophages expressed a low accessory activity.     

Characterization of protein Ser/Thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilin-cyclosporin complex.

Two major protein phosphatase (PP) activities were purified from cytosolic extracts of the erythrocytic stage of the malaria parasite, Plasmodium falciparum. Both enzymes were specific for phosphoserine and phosphothreonine residues with very little activity against phosphotyrosine residues. The biochemical properties of the enzymes suggested their strong similarity with eukaryotic PP2A and PP2B protein phosphatases. Both enzymes preferentially dephosphorylated the alpha subunit of phosphorylase kinase, and were resistant to inhibitor-1. The PP2A-like enzyme required Mn2+ for activity and was inhibited by nanomolar concentrations of okadaic acid (OA). The cDNA sequence of the PP2A-like enzyme was identified through a match of its predicted amino acid sequence with the N-terminal sequence of the catalytic subunit. The PP2B-like (calcineurin) enzyme was stimulated by calmodulin and Ca2+ or Ni2+, but was resistant to OA. Malarial calcineurin was strongly and specifically inhibited by cyclosporin A (CsA) only in the presence of wild type P. falciparum cyclophilin but not a mutant cyclophilin. The inhibition was noncompetitive, and provides a potential explanation for the cyclosporin-sensitivity of the parasite. There was no significant quantitative difference in the total protein Ser/Thr phosphatase activity among the ring, trophozoite, and schizont stages.     

The war between the malaria parasite and the immune system: immunity,immunoregulation and immunopathology

Throughout history malaria has proved to be a significant threat to human health. Between 300 and 500 million clinical cases occur each year worldwide, approximately 2 million of which are fatal, primarily in children. The vast majority of malaria‐related deaths are due to infection with Plasmodium falciparum; P. vivax causes severe febrile illness but is rarely fatal. Following repeated exposure to infection, people living in malaria endemic areas gradually acquire mechanisms to limit the inflammatory response to the parasite that causes the acute febrile symptoms (clinical immunity) as well as mechanisms to kill parasites or inhibit parasite replication (antiparasite immunity). Children, who have yet to develop protective immune mechanisms are thus at greater risk of clinical malaria, severe disease and death than adults. However, two epidemiological observations indicate that this is, perhaps, an oversimplified model. Firstly, cerebral malaria – a common manifestation of severe malaria – typically occurs in children who have already acquired a significant degree of antimalarial immunity, as evidenced by lower mean parasite densities and resistance to severe anaemia. One potential explanation is that cerebral malaria is, in part, an immune‐mediated disease in which immunological priming occurs during first infection, eventually leading to immunopathology on re‐infection. Secondly, among travelers from nonendemic areas, severe malaria is more common – and death rates are higher – in adults than in children. If severe malaria is an immune‐mediated disease, what might be priming the immune system of adults from nonendemic areas to cause immunopathology during their first malaria infection, and how do adults from endemic areas avoid severe immunopathology? In this review we consider the role of innate and adaptive immune responses in terms of (i) protection from clinical malaria (ii) their potential role in immunopathology and (iii) the subsequent development of clinical immunity. We conclude by proposing a model of antimalarial immunity which integrates both the immunological and epidemiological data collected to date.     

Cysteine-rich antimicrobial peptides of the cattle tick Boophilus microplus: isolation, structural characterization and tissue expression profile

Antimicrobial peptides (AMPs) are components of the immune system of both vertebrate and invertebrate animals. This study describes the isolation, primary structure, cDNA cloning, and tissue expression profile of two cysteine-rich AMPs from the hemolymph of the cattle tick Boophilus microplus. A 10,204 Da polypeptide, with six cysteine residues and no sequence similarity to any known molecule, was isolated from the cell-free hemolymph. Because of its sequence originality, this peptide was named microplusin. The second AMP was isolated from the hemocytes of B. microplus. This peptide, with a molecular mass of 4285 Da and six cysteines, is a defensin with similarity to the insect defensin family members. The cDNA cloning established that microplusin is synthesized as a prepeptide while the tick defensin is synthesized as a prepromolecule. Interestingly, despite the fact that microplusin and defensin have been isolated from different compartments, their gene expression was found to have similar tissue distribution.