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Biomedical subjects

G Langsley

Publications and source records attributed to G Langsley.

At least 19 recordsLinked to original sources
PubMed2001

TaCRK3 encodes a novel Theileria annulata protein kinase with motifs characteristic of the family of eukaryotic cyclin dependent kinases: a comparative analysis of its expression with TaCRK2 during the parasite life cycle.

The TaCRK3 gene from the bovine apicomplexan parasite Theileria annulata, encodes a 46 kDa polypeptide with strong homology to the eukaryotic family of cyclin-dependent kinases. TaCRK3 does not show significant alignment with any particular CDK group, other than the Pfmrk kinases from the related apicomplexans Plasmodium falciparum and Plasmodium yoelii. It has a putative bipartite nuclear localization signal and is located to parasite nuclei by IFAT. Protein levels are constitutive throughout differentiation of the intra-lymphocytic macroschizont. This contrasts with the expression pattern of TaCRK2 (Kinnaird et al., 1996, Mol. Microbiol., 22, 293-302) which is closely related to the eukaryotic CDK1 /2 families involved in regulation of cell cycle progression. TaCRK2 is also located to the parasite nuclei but has no nuclear localization signal and exhibits transient up-regulation in protein levels during mid-merogony. However compared to TaCRK3, it shows down-regulation near the end of merogony. We predict that TaCRK3 may have a role in regulation of gene transcription while TaCRK2 is more likely to be involved in control of parasite nuclear division.

Amino Acid Motifs↗
PubMed2001

The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.

Amino Acid Sequence↗
PubMed2000

Loss of matrix metalloproteinase 9 activity in Theileria annulata-attenuated cells is at the transcriptional level and is associated with differentially expressed AP-1 species.

The schizont stage of the protozoan parasite Theileria annulata reversibly transforms bovine monocytes into an immortalised and metastatic state. We have been studying T. annulata induction of host matrix metalloproteinases (MMP) which are involved in parasite dissemination and pathogenesis. We have observed that prolonged in vitro culture of T. annulata-infected cell lines results in their attenuation and this process is associated with alterations in both host and parasite gene expression. In particular, a loss in bovine MMP expression in later passage cultures suggests that these parasite-induced MMPs are virulence factors. As a means to further our understanding of the attenuation process we examine in detail the parasite-induced differential expression of one particular bovine proteinase, MMP9, in non-attenuated (p58) and attenuated (p158) passage levels of the Ode vaccine line. We show here that MMP9 expression is regulated at the transcriptional level and we suggest that a particular parasite-induced AP-1 recognition transcription factor present in the Ode non-attenuated line may have a role to play in the expression of this host gene.

Animals↗
PubMed2000

A Theileria parva type 1 protein phosphatase activity.

The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1.

Animals↗
PubMed2000

Constitutive PI3-K activity is essential for proliferation, but not survival, of Theileria parva-transformed B cells.

Theileria is an intracellular parasite that causes lymphoproliferative disorders in cattle, and infection of leucocytes induces a transformed phenotype similar to tumour cells, but the mechanisms by which the parasite induces this phenotype are not understood. Here, we show that infected B lymphocytes display constitutive phosphoinositide 3-kinase (PI3-K) activity, which appears to be necessary for proliferation, but not survival. Importantly, we demonstrate that one mechanism by which PI3-K mediates the proliferation of infected B lymphocytes is through the induction of a granulocyte-monocyte colony-stimulating factor (GM-CSF)-dependent autocrine loop. PI3-K induction of GM-CSF appears to be at the transcriptional level and, consistently, we demonstrate that PI3-K is also involved in the constitutive induction of AP-1 and NF-kappaB, which characterizes Theileria-infected leucocytes. Taken together, our results highlight a novel strategy exploited by the intracellular parasite Theileria to induce continued proliferation of its host leucocyte.

Animals↗
PubMed2000

Plasmodium falciparum rab6 GTPase: expression, purification, crystallization and preliminary crystallographic studies.

The Plasmodium falciparum rab6 gene encodes a 208 amino-acid polypeptide. Two recombinant versions of P. falciparum Rab6 protein were expressed in Escherichia coli: the full-length protein and a truncated form containing residues 1-175. Both forms were purified from the soluble fraction of bacterial extract and were purified by ion-exchange chromatography and size-exclusion chromatography. Purified proteins were crystallized at pH 6.5 using the hanging-drop vapor-diffusion technique at room temperature. The full-length protein diffracted to 2.4 A and belongs to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 80. 6, c = 90.4 A. The crystals of the truncated protein were isomorphous with those of the full-length construct and diffracted X-rays to 2.2 A resolution.

Animals↗
PubMed2000

Structure of the nucleotide-binding domain of Plasmodium falciparum rab6 in the GDP-bound form.

Rab proteins are small Ras-like GTPases which play important roles in regulating intracellular vesicle trafficking. The nucleotide-binding domain of Rab6 from the malaria parasite Plasmodium falciparum was crystallized with GDP bound to the active site. The MAD phasing technique was used to determine the crystal structure to 2.3 A resolution. Comparisons of the structure of GDP-bound PfRab6 with the recently determined structures of Rab3A in complex with either a GTP analog or with GTP and Rabphillin present structural evidence supporting the traditional model for the molecular GTP/GDP switch in Rab proteins. PfRab6 residues homologous to those distinguishing human Rab6 isoforms, which differ in binding to Rabkinesin-6 in human cells, are located next to the recognized complementarity-determining region (CDR) and constitute a conceptual broadening of that domain. Despite significant observable differences in Golgi ultrastructure, the Rab6 core structure and switch mechanism appear highly conserved when compared with murine Rab3a structures. A significant difference between the PfRab6 and higher eukaryotic Rabs may be the lack of CDR features that allow binding interactions with Rabkinesin-type effectors.

Amino Acid Sequence↗
PubMed1999

Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1-42 maturase.

The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2's subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.

Amino Acid Sequence↗
PubMed1999

Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome.

The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated'). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic gamma-tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.

Amino Acid Sequence↗
PubMed1999

Novel secretory pathways in Plasmodium?

The secretion of proteins from intraerythrocytic stages of Plasmodium falciparum into the infected host cell is still poorly understood. A recent proposal that two distinct, mutually exclusive, secretory compartments may exist within the parasite cell has received much attention. Denise Mattei, Gary Ward, Gordon Langsley and Klaus Lingelbach here critically discuss the data on which this model is based, and then they address a more general question: to what extent are unusual aspects of protein secretion in Plasmodium unique among eukaryotic cells?

Animals↗
PubMed1999

The use of nocodazole in cell cycle analysis and parasite purification from Theileria parva-infected B cells.

Theileria parasites transform bovine leukocytes and induce uncontrolled lymphoproliferation only in the macroschizont stage of their life cycle. The isolation of highly purified stage-specific parasite RNA and proteins is an essential prerequisite when studying the Theileria-host relationship. We therefore improved a protocol based on the cytolytic bacterial toxin aerolysin by taking advantage of the microtubule inhibitor nocodazole. In this report we describe that nocodazole-mediated separation of the parasite from the host cell microtubule network was used with success to improve quantity and quality of purified parasites. We furthermore show that nocodazole is a useful tool to study cell cycle checkpoints due to its capacity to induce reversible cell cycle arrest in Theileria-infected B cells.

Animals↗
PubMed1999

Theileria annulata in CD5(+) macrophages and B1 B cells.

Theileria parasites infect and transform bovine leukocytes. We have analyzed laboratory-established Theileria sp.-infected leukocyte lines and observed that transformed macrophages express CD5. Low-level expression of CD5 by macrophages was further confirmed on three independent Theileria annulata clinical isolates from Tunisia. Interestingly, the fourth CD5(+) clinical isolate (MB2) was morphologically different, expressed surface immunoglobulin M (IgM) and BoLA class II, and had rearranged Ig light-chain genes. To demonstrate that MB2 did indeed contain CD5(+) B cells, individual clonal lines were obtained by limiting dilution, and CD5 expression and Ig gene rearrangement were confirmed. This suggests that in natural infections T. annulata can invade and transform CD5(+) B cells.

Animals↗
PubMed1998

A Plasmodium falciparum novel gene encoding a coronin-like protein which associates with actin filaments.

Plasmodium falciparum, the major causative agent of human malaria, is an Apicomplexa protozoan parasite which invades in its intermediate host hepatocytes and erythrocytes. The driving force underlying internalization into the host cell is thought to involve both polymerization of parasite actin, as entry is inhibited by the cytochalasins, and an actin motor-associated protein. In the related Apicomplexa parasite, Toxoplasma gondii, the involvement of parasite actin during both processes of motility and host cell entry has been genetically established. In a search for molecules that can regulate actin dynamics within Apicomplexa parasites, we have identified a P. falciparum homologue of the actin associated protein called coronin originally described in the amoeba Dictyostelium discoideum. The single copy gene displays a strong homology with the amoeba sequence and with the bovine and human coronin homologues recently cloned. This homology lies not only within the N-terminus containing the five WD repeats that characterize coronin but also extends in the C-terminal part. Furthermore, using an affinity-purified mouse monoclonal antibody against D. discoideum coronin, we have detected in extracts of P. falciparum young and mature schizonts a 42-kDa polypeptide which binds this antibody and is present in a Triton insoluble fraction that also contains parasite actin filaments. In addition, the recombinant protein encoded by the homologue nucleotidic sequence of P. falciparum coronin is indeed recognized by the antibody against D. discoideum coronin. Finally, the cross-reactive polypeptide displays the ability to cosediment with exogenous F-actin, a property which fits with its involvement in actin dynamics.

Actins↗
PubMed1998

Upregulation of Jun and Fos family members and permanent JNK activity lead to constitutive AP-1 activation in Theileria-transformed leukocytes.

Theileria parasitises bovine leukocytes and transforms them into proliferating, metastatic tumours, where the infection resembles a leukaemia-like disease. We have studied the signal transduction pathways leading to activation of the transcription factor AP-1 in different transformed leukocytes. Parasite infection leads to an up-regulation of all members of the Jun/Fos family of proteins and surprisingly, this occurs in the absence of any detectable ERK, or p38 MAP kinase activity. In the parasitised B-sarcoma TBL3, AP-1 induction occurs in the absence of any JNK activity. In contrast, in infected macrophage and B-cell lines, AP-1 transcriptional activity is strictly associated with the parasite-induced constitutive activation of JNK and subsequent c-Jun N-terminal phosphorylation. Thus, constant AP-1 transcriptional activity involves both an upregulation in the levels of Jun and Fos proteins and constitutive JNK activation.

Animals↗
PubMed1998

A primary malarial infection is composed of a very wide range of genetically diverse but related parasites.

To address the question of how many distinct parasites are injected when a mosquito bites, we have characterized isolates resulting most probably from a single sporozoite inoculum. We describe the direct and immediate cloning on hepatocyte feeder layers of a Thai and an African Plasmodium falciparum primary isolate and the characterization of 67 independent clones by four techniques totaling nine different markers. This led to three main conclusions: (a) both the phenotypic and genotypic markers revealed an unexpectedly large degree of diversity within the clones from a single isolate; (b) the clones are nonetheless genetically related; and (c) a single mosquito inoculum would most likely be sufficient to generate considerable isolate complexity in the absence of repeated exposure. This diversity, which has been greatly underestimated in previous studies, does not bode well for the development of successful malaria control means.

Africa↗
PubMed1997

Plasmodium falciparum asparagine and aspartate rich protein 2 is an evolutionary conserved protein whose repeats identify a new family of parasite antigens.

We describe here a new Plasmodium falciparum antigen, asparagine and aspartate rich protein 2 (PfAARP2) of 150 kDa, which is encoded by a unique gene on chromosome 1. PfAARP2 is first expressed 12 h post-invasion and accumulates in trophozoites and schizonts. Immunofluorescence studies indicate that PfAARP2 is translocated into the red blood cell cytoplasm. The central region of Pfaarp2 contains blocks of repetitions encoding asparagine and aspartate residues, which define a new family of related genes dispersed on different chromosomes, and two members of this family have also been identified. Interestingly, the non-repeated N- and C-termini of PfAARP2 display significant similarity to two yeast and human predicted proteins, and its possible function is discussed.

Amino Acid Sequence↗
PubMed1997

Plasmodium falciparum AARP1, a giant protein containing repeated motifs rich in asparagine and aspartate residues, is associated with the infected erythrocyte membrane.

During Plasmodium falciparum asexual intraerythrocytic development, the host's cell plasma membrane is modified by the insertion of parasite proteins. One or more of these modifications mediate the cytoadherence of infected erythrocytes to host vascular endothelium. However, these surface antigens can be the target of cytophilic antibodies which promote phagocytosis of the infected erythrocyte. It has been proposed that antibodies directed to epitopes rich in asparagine play an important role in this process, which has promoted efforts to isolate the corresponding gene(s). We describe here P. falciparum asparagine- and aspartate-rich protein 1 (PfAARP1), a new giant (circa 700-kDa) protein associated with the infected erythrocyte membrane which is rich in asparagine and aspartate residues due to the presence of nine blocks of repeats. Topology analysis predicts that PfAARP1 has multiple transmembrane domains and at least five external loops. Human antibodies immunopurified against a sequence composed exclusively of asparagine and aspartate amino acids derived from PfAARP1 label the surface of the infected erythrocyte, demonstrating that such motifs are exposed. Interestingly, external loop 4 of PfAARP1 contains repetitions of these residues, and their possible role as a target of cytophilic antibodies is discussed.

Amino Acid Sequence↗