The vertical ramus osteotomy for high condylar fractures.
Explore the source record for details and available documents.
Adult↗
Biomedical subjects
D Chakrabarti
Publications and source records attributed to D Chakrabarti.
Characterization of an eukaryotic peptide deformylase from Plasmodium falciparum.
Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Until recently, PDF has been thought as an enzyme unique to the bacterial kingdom. Searches of the genomic DNA databases identified several genes that encode proteins of high sequence homology to bacterial PDF from eukaryotic organisms. The cDNA encoding Plasmodium falciparum PDF (PfPDF) has been cloned and overexpressed in Escherichia coli. The recombinant protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is inhibited by specific PDF inhibitors. Western blot analysis indicated expression of mature PfPDF in trophozoite, schizont, and segmenter stages of intraerythrocytic development. These results provide strong evidence that a functional PDF is present in P. falciparum. In addition, PDF inhibitors inhibited the growth of P. falciparum in the intraerythrocytic culture.
Amidohydrolases↗
Characterization of a novel serine/threonine protein phosphatase (PfPPJ) from the malaria parasite, Plasmodium falciparum.
A novel protein phosphatase cDNA of the PPP superfamily was identified from the malaria parasite, Plasmodium falciparum (Pf), and tentatively named PfPPJ. The predicted primary structure of the phosphatase contained all the known conserved motifs of the PPP superfamily essential for catalytic activity. The enzyme was specific for dephosphorylation of phosphoserine and phosphothreonine residues with very little activity against phosphotyrosine residues. However, the sequence at its C-terminal end was unique, and was consistent with its resistance to the classical PP2A-specific inhibitors such as okadaic acid and microcystin-LR, and the PP1-specific inhibitor, mammalian heat-stable inhibitor-2 (I-2). Even the catalytic core of PfPPJ had a sequence substantially different from the other PPPs such that PfPPJ could be placed in an apparently separate phylogenetic branch. At 294 amino acids residues, PfPPJ was one of the smallest okadaic acid-resistant PPP phosphatases known. By Northern blot analysis, the expression of the PfPPJ mRNA showed the following pattern: schizont > ring > trophozoite, which closely paralleled the expression of the protein, as determined by immunofluorescence. Together, these results suggested a parasitic stage-specific transcriptional regulation of this novel and potentially unique protozoan phosphatase.
Amino Acid Sequence↗
Co-ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis.
Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.
Animals↗
PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum.
We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T(173)PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn(2+) over Mg(2+) ions as a cofactor. The Lys(38)-->Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.
Alternative Splicing↗
Activation of a Plasmodium falciparum cdc2-related kinase by heterologous p25 and cyclin H. Functional characterization of a P. falciparum cyclin homologue.
Several Plasmodium falciparum genes encoding cdc2-related protein kinases have been identified, but the modalities of their regulation remains largely unexplored. In the present study, we investigated the regulation in vitro of PfPK5, a putative homologue of Cdk1 (cdc2) in P. falciparum. We show that (i) PfPK5 is efficiently activated by heterologous (human) cyclin H and p25, a cyclin-like molecule that specifically activates human Cdk5; (ii) the activated enzyme can be inhibited by chemical Cdk inhibitors; (iii) Pfmrk, a putative P. falciparum homologue of the Cdk-activating kinase, does neither activate nor phosphorylate PfPK5; and (iv) PfPK5 is able to autophosphorylate in the presence of a cyclin. Taken together, these results suggest that the regulation of Plasmodium Cdks may differ in important aspects from that of their human counterparts. Furthermore, we cloned an open reading frame encoding a novel P. falciparum protein possessing maximal homology to cyclin H from various organisms, and we show that this protein, called Pfcyc-1, is able to activate recombinant PfPK5 in vitro with an efficiency similar to that of human cyclin H and p25. This work opens the way to the development of screening procedures aimed at identifying compounds that specifically target the parasite Cdks.
Amino Acid Sequence↗
The cell cycle in protozoan parasites.
Research into cell cycle control in protozoan parasites, which are responsible for major public health problems in the developing world, has been hampered by the difficulties in performing classical genetic analysis with these organisms. Nevertheless, in a large part thanks to the data gathered in other eukaryotic systems and to the acquisition of the sequences of parasite genes homologous to cell cycle regulators, many molecular tools required for an in-depth study of the cell cycle in protozoan parasites have been collected over the past few years. Despite the considerable phylogenetic divergence between these organisms and other eukaryotes, and notwithstanding important specificities such as the apparent lack of checkpoints during cell cycle progression, available data indicate that the major families of cell cycle regulators appear to operate in protozoan parasites. Functional studies are now needed to define the precise role of these regulators in the life cycle of the parasites, and to possibly validate cell cycle control elements as potential targets for chemotherapy.
Animals↗
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.
Amino Acid Sequence↗
An RCC1-type guanidine exchange factor for the Ran G protein is found in the Plasmodium falciparum nucleus.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Protein prenyl transferase activities of Plasmodium falciparum.
Prenylated proteins have been shown to function in important cellular regulatory processes including signal transduction. The enzymes involved in protein prenylation, farnesyl transferase and geranylgeranyl transferase, have been recent targets for development of cancer chemotherapeutics. We have initiated a systematic study of protein prenyl transferases of the malaria parasite, Plasmodium falciparum, to determine whether these enzymes can be developed as targets for antimalarial chemotherapy. We report here the identification of protein farnesyl transferase and protein geranylgeranyl transferase-I in the malaria parasite, P. falciparum. The farnesyl transferase has been partially purified from the cytosolic fraction through ammonium sulfate precipitation and Mono-Q chromatography. Farnesyl and geranylgeranyl transferase-I activities are present at all stages of P. falciparum intraerythrocytic development with maximum specific activity in the ring stage. Geranylgeranyl transferase-I specific activity is two times that of farnesyl transferase in the ring stage. Peptidomimetics and prenyl analogues of protein farnesyl transferase substrates were tested as in vitro inhibitors of partially purified P. falciparum prenyl transferase and of malaria parasite growth. The peptidomimetics were significantly more potent inhibitors than lipid substrate analogues of both the activity of Mono-Q purified enzyme and parasite growth in intraerythrocytic cultures. Exposure of the parasite to the peptidomimetic L-745,631 also showed significant inhibition of morphological development beyond the trophozoite stage. These studies suggest the potential of designing or identifying differential inhibitors of P. falciparum and mammalian prenyl transferases as an approach to novel malaria therapy.
Alkyl and Aryl Transferases↗
Evidence for the shikimate pathway in apicomplexan parasites.
Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.
3-Phosphoshikimate 1-Carboxyvinyltransferase↗
Antimalarial activities of polyhydroxyphenyl and hydroxamic acid derivatives.
Several known mammalian ribonucleotide reductase inhibitors featuring a polyhydroxyphenyl and/or hydroxamate moiety as the active group were screened for potency in inhibiting growth of the malaria parasite Plasmodium falciparum. Compounds containing a 2,3- or 3,4-dihydroxyphenyl group as well as benzohydroxamate appear to be the most effective inhibitors of the malaria parasite.
Animals↗
Identification, cloning, and mutational analysis of the casein kinase 1 cDNA of the malaria parasite, Plasmodium falciparum. Stage-specific expression of the gene.
The cDNA for casein kinase 1 (CK1) of Plasmodium falciparum was cloned, sequenced, and expressed in bacteria. The single major open reading frame of the 1.2-kilobase pair cDNA coded for a 324-amino acid polypeptide of approximately 37 kDa, the predicted sequence of which showed strong identity with known CK1 isoforms. The purified recombinant enzyme exhibited properties characteristic of CK1, such as inhibition by CK1-7, the ability to phosphorylate a highly specific peptide substrate, and a strong preference for ATP over GTP. A casein kinase activity, partially purified from soluble extracts of P. falciparum by affinity chromatography through CK1-7 columns displayed identical properties. The activity showed a stage-specific expression in the parasite, in the order trophozoite > ring >> schizont. Northern analysis indicated the existence of two major CK1 mRNAs, 2.4 and 3.2 kilobase pairs long, the levels of which were in the order ring > schizont > trophozoite. Mutagenesis of recombinant CK1 defined important amino acid residues and their potential role in the conformation of the enzyme. The malarial CK1 appeared to be the one of the smallest and perhaps the most primitive CK1 enzymes known, containing little sequence information beyond the minimal catalytic domain.
Amino Acid Sequence↗
Cloning and characterization of Plasmodium falciparum ADP-ribosylation factor and factor-like genes.
Explore the source record for details and available documents.
ADP-Ribosylation Factors↗
IFN-alpha induces autoimmune T cells through the induction of intracellular adhesion molecule-1 and B7.2.
Insulin-dependent diabetes is an autoimmune disease characterized by the loss of the insulin-producing beta cells and the appearance of autoreactive (anti-islet) T cells. The mechanism by which these autoimmune T cells become activated has not been resolved. We demonstrate that the expression of IFN-alpha by the pancreatic beta cells leads to the development of CD4+ T cells that proliferate in the presence of islet Ags. These autoreactive T cells have a Th1 phenotype and are able to lyse islet cells, possibly through an indirect, cytokine-mediated mechanism. We also demonstrate that the pancreatic infiltrating leukocytes in the transgenic mice express increased levels of 87.2 and ICAM-1 and that IFN-alpha can directly induce these two costimulatory molecules on nontransgenic splenic APCs. Treatment of the transgenic mice with Abs against these costimulatory molecules demonstrated that B7.2 is essential for the induction of autoreactive T cells, whereas ICAM-1 contributes to but is not essential for the formation of these autoreactive cells. As B7.2 and ICAM-1 are able to synergize to provide costimulatory signals to naive T cells under conditions of limiting Ag, we propose that IFN-alpha expression normally contributes to the development of an anti-viral cellular immune response, but that uncontrolled expression of this cytokine will induce autoimmunity.
Animals↗
Plasmodium falciparum: the small GTPase rab11.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Current status of the Plasmodium falciparum genome project.
The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures. Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasite's genes and to prepare an ordered set of overlapping clones spanning each of the parasite's 14 chromosomes. Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasite's genes, and approximately 5% of the parasite's genes are tentatively identified from similarity searches of entries in the international sequence databases. A total of > 0.5 Mb of P. falciparum sequence tag data is available. The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome. Routes of access to project information and services are described.
Animals↗
Identification of a family of Rab G-proteins in Plasmodium falciparum and a detailed characterisation of pfrab6.
As a first step towards developing a set of compartment-specific probes for studying protein trafficking in the malaria-infected erythrocyte, we describe here a family of Plasmodium falciparum Rab proteins. We characterise in detail P. falciparum Rab6 (PfRab6) a marker which in other cells is specific for the Golgi/trans Golgi network. Although PfRab6 mRNA is expressed throughout the intraerythrocytic cycle, maximal expression occurs at the trophozoite stage. Immunofluorescence microscopy shows that the distribution of PfRab6 changes during the final stages of parasite maturation, coalescing into multiple foci, each of which is associated with the nucleus of a forming daughter parasite.
Amino Acid Sequence↗