Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine and tyrosine and O-glycosylated in Trichomonas vaginalis

The eukaryotic translation factor eIF-5A is highly conserved throughout eukaryotes and undergoes an unusual polyamine-dependent post-translational modification called hypusination. Trichomonas vaginalis has two tveif-5a genes (tveif-5a1 and tveif-5a2), each encoding a 19-kDa protein. In this report, we describe the detection of two forms with different isoelectric points (5.2 and 5.5) that correspond to the precursor and mature TveIF-5A, respectively. In addition, we demonstrated that only the mature form of TveIF-5A is phosphorylated and glycosylated via two-dimensional gel electrophoresis-western blot (2DE-WB) assays using anti-phosphoserine and anti-phosphotyrosine antibodies and the SNA, ConA and MAA lectins. Interestingly, when the protozoa were grown in 1,4-diamino-2-butanone (DAB), an inhibitor of putrescine biosynthesis, and transferred to medium containing exogenous putrescine, a new spot with an isoelectric point of 5.3 was observed, presumably corresponding to a phosphorylated intermediate or deoxyhypusine form. Our data indicate that, in T. vaginalis, phosphorylations and glycosylations are necessary to obtain the mature TveIF-5A, and we confirm the identity of the precursor, intermediate and mature forms of TveIF-5A by mass spectrometry analysis.     

Interspersed repetitive DNA from Plasmodium falciparum

An interspersed repetitive DNA element from the Camp strain of the human malaria parasite Plasmodium falciparum was cloned and sequenced. The element is repeated at least 11 times in the genome, is a minimum of 2.0 kilobases long and maps to eight or more different parasite chromosomes. The cloned DNA includes a 1.0 kilobase open reading frame. RNA complementary to the repetitive element is found within blood stage forms of the parasite, but only in the most mature forms. Searches of DNA and protein sequence databases for homology to the element were unsuccessful. Sizes of restriction enzyme fragments which hybridize to the element show considerable variation between strains, indicating that the repeat family represented by this element could be transposable.     

Translation initiation factor eIF-4gamma is encoded by an amplified gene and induces an immune response in squamous cell lung carcinoma

Amplification of cellular oncogenes is an important mechanism of altered gene expression in human cancers. Using comparative genomic hybridization we recently identified an amplification at 3q26.1-q26.3 in 30% of squamous cell carcinomas of the lung. A variety of methods including microdissection-mediated procedures permit cloning of genes encoded within amplified domains but do not directly lead to the identification of biologically relevant genes. In this study, we have circumvented this problem by combining an immunological and molecular genetic approach to analyze squamous cell lung carcinoma. To identify both amplified and tumor relevant genes, we generated a cDNA expression library from a tumor with the 3q amplification and hybridized the expressed recombinant polypeptides with the autologous serum. Of 400000 cDNA clones we identified 17 antigens which induce an immune response in a patient with squamous cell lung carcinoma. While most clones represent individual genes sequence analysis revealed that four of the 17 cDNAs are nearly identical with the eukaryotic translation initiation factor (eIF)-4gamma recently assigned on 3q. We demonstrated that the gene for eIF-4gamma was amplified within 3q26-q27 in independent squamous cell lung carcinomas. In this study, we report the identification of several antigens which elicit an immune response in a squamous cell lung carcinoma patient including eIF-4gamma. eIF-4gamma is encoded by an amplified gene and possibly plays a crucial part in the development of squamous cell lung carcinoma.      

Identification and biochemical characterisation of a protein phosphatase 5 homologue from Plasmodium falciparum

We report the identification of a new serine/threonine phosphatase from Plasmodium falciparum at the DNA and protein levels. A 1.8 kb cDNA fragment encoding the protein phosphatase was identified via PCR amplification. The sequence has a coding capacity of 594 amino acids. Immunoblot analysis of P. falciparum extracts showed that antibodies generated against the His₆-fusion protein recognise a protein of approximately 80 kDa. The deduced amino acid sequence shares 55% identity with a mouse protein, identified as Protein Phosphatase 5 (PP5). We show that the P. falciparum PP5 homologue (PfPP5) has all structural and functional characteristics of this class of enzymes. It contains three tetratricopeptide repeats (TPR) and a nuclear targeting sequence at its N-terminus and a highly conserved C-terminal catalytic domain. Southern blot results are compatible with the existence of PfPP5 as a single copy gene. Purified recombinant protein, like the native protein enriched from P. falciparum extracts exhibited phosphatase activity that can be enhanced by both arachidonic and oleic acids, but not by myristic or stearic acid. In addition, the activity is inhibited by okadaic acid (OA) with an IC₅₀ of 4 nM. Immunofluorescence microscopy has localised PfPP5 preferentially to the nucleus. The function of PfPP5 is presently unclear, but like other PP5s of many eukaryotic organisms, it may have important regulatory functions in the parasite cell cycle.     

Peptidases from Plasmodium falciparum cultured in vitro

An acid peptidase that degrades hemoglobin optimally at pH 3.5, a neutral aminopeptidase and an alkaline endopeptidase that acts on an alpha-N-blocked synthetic substrate have been demonstrated in Plasmodium falciparum in culture. The enzymes were shown to be distinct by anion exchange chromatography, gel filtration on Sephadex G-200 and isoelectric focusing. The activities of the acid peptidase and the aminopeptidase were inhibited by antimalarial compounds.     

Tumor necrosis factor enhances neutrophil-mediated killing of Plasmodium falciparum.

We developed a radiometric assay by which the antiplasmodial effects of phagocytic cells can be quantitated. This assay was used to examine the effects of recombinant human tumor necrosis factor alpha (TNF-alpha) on the killing of Plasmodium falciparum by human neutrophils. Data presented demonstrated that neutrophils engulf and destroy P. falciparum, but substantial killing of parasites required the presence of either heat-labile or heat-stable opsonins. While recombinant TNF-alpha at concentrations of 5 to 50,000 U/ml showed no direct effects on the parasite, this cytokine augmented the antimalarial activity of neutrophils at doses of 20 to 250 U/10(6) neutrophils. The results suggest that TNF-alpha is an important component of the immune phagocytic effector mechanisms which are involved in destruction of the malarial parasite.     

A novel dual Dbp5/DDX19 homologue from Plasmodium falciparum requires Q motif for activity

Helicases are ubiquitous essential enzymes which have significant role in the nucleic acid metabolism. Using in silico approaches in the recent past we have identified a number of helicases in the Plasmodium falciparum genome. In the present study we report purification and detailed characterization of a novel helicase from P. falciparum. Our results indicate that this helicase is a homologue of Dbp5 and DDX19 from yeast and human, respectively. The biochemical characterization shows that it contains DNA and RNA unwinding, nucleic acid dependent ATPase and RNA binding activities. It is interesting to note that this enzyme can unwind DNA duplexes in both 5' to 3' and 3' to 5' directions. Using truncated derivatives we further show that Q motif is essentially required for all of its activities. These studies should make an important contribution in understanding the enzymes involved in nucleic acid metabolism in the parasite.     

Two soluble antigens of Plasmodium falciparum induce tumor necrosis factor release from macrophages.

The production of cytokines such as tumor necrosis factor (TNF) may contribute to the pathology of malaria. We showed previously that crude preparations of heat-stable exoantigens from parasite cultures induce the release of TNF in vitro and in vivo. When separated from the culture medium by affinity chromatography, in which immune immunoglobulin G was used as ligand, the mixture of exoantigens of Plasmodium falciparum retained the capacity to induce the secretion of TNF, both by human monocytes from Gambian children and by mouse macrophages. Two individual antigens, Ag1 and Ag7, further purified by affinity chromatography and identified by crossed immunoelectrophoresis, also stimulated TNF production by both types of cell but differed in other functional properties. Thus, the activity of Ag7, but not that of Ag1, was inhibited by polymyxin B, and antisera made against boiled exoantigens of the rodent parasite Plasmodium yoelii which blocked the ability of these antigens to induce the production of TNF also inhibited the activity of Ag7 without affecting Ag1. Since the prevalence of antibody against Ag7 in sera from children in endemic areas appears to correlate with the development of immunity against the manifestations of the disease, this antigen may be one cause of pathology, perhaps through its ability to induce the production of TNF. Its serological relationship with rodent exoantigens suggests that it might be a candidate for an anti-disease vaccine which has the advantage that its active moiety is not subject to significant antigen polymorphism.     

A cysteine protease activity from Plasmodium falciparum cleaves human erythrocyte ankyrin

The malaria parasite Plasmodium falciparum undergoes distinct morphologic changes during its 48-h life cycle inside human red blood cells. Parasite proteinases appear to play important roles at all stages of the erythrocytic cycle of human malaria. Proteases involved in erythrocyte rupture and invasion are possibly required to breakdown erythrocyte membrane skeleton. To identify such proteases, soluble cytosolic extract of isolated trophozoites/schizonts was incubated with erythrocyte membrane ghosts or spectrin-actin depleted inside-out vesicles, which were then analyzed by SDS-PAGE. In both cases, a new protein band of 155 kDa was detected. The N-terminal peptide sequencing established that the 155 kDa band represents truncated ankyrin. Immunoblot analysis using defined monoclonal antibodies confirmed that ankyrin was cleaved at the C-terminus. While the enzyme preferentially cleaved ankyrin, degradation of protein 4.1 was also observed at high concentrations of the enzyme. The optimal activity of the purified enzyme, using ankyrin as substrate, was observed at pH 7.0-7.5, and the activity was strongly inhibited by standard inhibitors of cysteine proteinases (cystatin, NEM, leupeptin, E-64 and MDL 28 170), but not by inhibitors of aspartic (pepstatin) or serine (PMSF, DFP) proteinases. Furthermore, we demonstrate that protease digestion of ankyrin substantially reduces its interaction with ankyrin-depleted membrane vesicles. Ektacytometric measurements showed a dramatic increase in the rate of fragmentation of ghosts after treatment with the protease. Although the role of ankyrin cleavage in vivo remains to be determined, based on our findings we postulate that the parasite-derived cysteine protease activity cleaves host ankyrin thus weakening the ankyrin-band 3 binding interactions and destabilizing the erythrocyte membrane skeleton, which, in turn, facilitates parasite release. Further characterization of the enzyme may lead to the development of novel antimalarial drugs.     

A role for rheumatoid factor enhancement of Plasmodium falciparum schizont inhibition in vitro.

Studies were undertaken to determine whether rheumatoid factor (RF) was present in immune human and Aotus trivirgatus monkey sera which inhibited Plasmodium falciparum schizonts in vitro and to determine whether RF could be responsible for or contribute to merozoite agglutination in the parasite inhibition test. Additional studies were conducted to determine the effect of exogenous RF on schizont inhibition when used alone or in conjunction with immune or normal sera. RF was not detected in any of the 11 immune monkey sera or the 3 immune human sera which were tested. However, when RF was added to immune human or Aotus sera, levels of schizont inhibition increased significantly over levels obtained with immune serum alone. When RF was used alone or in conjunction with normal sera, levels of schizont inhibition were comparable to those obtained with normal serum. Furthermore, adsorption of the RF with immunoglobulin G-coated erythrocytes removed the enhancing activity. The results of this study indicate that RF, which is sometimes produced during acute or chronic malarial infection, may contribute nonspecifically to the enhanced clearance of plasmodia in vivo.     

Tumor necrosis factor does not induce Plasmodium falciparum crisis forms.

Mouse and rabbit sera from animals treated with Mycobacterium bovis BCG and lipopolysaccharide contained tumor necrosis factor (TNF) and induced malaria parasite crisis forms. However, neither purified mouse- nor recombinant DNA-produced human TNF induced crisis forms in cultured Plasmodium falciparum. Furthermore, rabbit polyclonal and mouse monoclonal antibodies against human TNF did not block the parasite inhibitory activity of human malaria crisis form factor serum from Sudan.     

Immunoglobulin E, a pathogenic factor in Plasmodium falciparum malaria.

Most children and adults living in areas where the endemicity of Plasmodium falciparum malaria is high have significantly elevated levels of both total immunoglobulin E (IgE) and IgE antimalarial antibodies in blood. This elevation is highest in patients with cerebral malaria, suggesting a pathogenic role for this immunoglobulin isotype. In this study, we show that IgE elevation may also be seen in severe malaria without cerebral involvement and parallels an elevation of tumor necrosis factor alpha (TNF). IgE-containing serum from malaria immune donors was added to tissue culture plates coated with rabbit anti-human IgE antibodies or with P. falciparum antigen. IgE-anti-IgE complexes as well as antigen-binding IgE antibodies induced TNF release from peripheral blood mononuclear cells (PBMC). Nonmalaria control sera with no IgE elevation induced significantly less of this cytokine, and the TNF-inducing capacity of malaria sera was also strongly reduced by passing them over anti-IgE Sepharose columns. The cells giving rise to TNF were adherent PBMC. The release of this cytokine probably reflects cross-linking of their low-affinity receptors for IgE (CD23) by IgE-containing immune complexes known to give rise to monocyte activation via the NO transduction pathway. In line with this, adherent monocytic cells exposed to IgE complexes displayed increased expression of CD23. As the malaria sera contained IgG anti-IgE antibodies, such complexes probably also play a role in the induction of TNF in vivo. Overproduction of TNF is considered a major pathogenic mechanism responsible for fever and tissue lesions in P. falciparum malaria. This overproduction is generally assumed to reflect a direct stimulation of effector cells by certain parasite-derived toxins. Our results suggest that IgE elevation constitutes yet another important mechanism involved in excessive TNF induction in this disease.     

Plasmodium falciparum biosynthesizes sulfoglycosphingolipids

Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active participants in adhesion processes in many systems and appear to be involved in the regulation of cell proliferation, differentiation and other developmental cellular events. However, the body of knowledge about synthesis, structure, and function of glycolipids in parasitic protozoa is very limited so far. In this work, we show by metabolic incorporation of [(14)C]palmitic acid, [(14)C]glucose and Na(2)(35)SO(4) that sulfoglycosphingolipids are biosynthesized in the three intraerythrocytic stages of Plasmodium falciparum. After saponification, purification of the labelled acidic components was achieved and two components named SPf1 and SPf2 were characterized. Chemical degradations and TLC analysis pointed out to sulfolipidic structures. Analysis by UV-MALDI-TOF mass spectrometry in the negative ion mode using nor-harmane as matrix showed for SPf2 a structure consisting in a disulfated hexose linked to a 20:1 sphingosine acylated with C18:0 fatty acid. Interestingly, parasite treatment with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) caused an arrest on parasite development associated to the inhibition of sulfoglycolipid biosynthesis. Taking into account that sulfoglycolipidic structures are currently involved in adhesion processes, our findings open the possibility to study the participation of this type of structures in the described aggregation phenomena in severe malaria and may contribute to clarify the pathogenesis of the disease. This report shows for the first time the synthesis of sulfoglycolipids in Apicomplexa.     

A fluorometric sensitivity test for chloroquine in Plasmodium falciparum isolates from patients

A new technique for assessment of in vitro growth of Plasmodium falciparum by fluoroassay was used to determine the sensitivity to chloroquine of parasites from cases of malaria imported into Japan. The technique was reliable, giving comparable results to the Rieckmann test, and is also applicable in the field.