RESUMO
We have analyzed the effect of antibodies (Abs) directed against major histocompatibility complex (MHC) class II Abs on the proliferation of Theileria parva-infected (Tpi) T cells. Anti-MHC class II Abs exert a direct effect on Tpi T cells causing an acute block in their proliferation. The inhibition does not involve apoptosis and is also entirely reversible. The rapid arrest of DNA synthesis caused by anti-MHC class II Abs is not due to interference with the state of activation of the T cells since the transcriptional activator NF-kappa B remains activated in arrested cells. In addition, interleukin 2 (IL-2), IL-2R, and c-myc gene expression are also unaffected. By analyzing the cell-cycle phase distribution of inhibited cells, it could be shown that cells in all phases of the cell cycle are inhibited. The signal transduction pathway that results in inhibition was shown to be independent of protein kinase C and extracellular Ca2+. Tyrosine kinase inhibitors, however, partly reduced the level of inhibition and, conversely, phosphatase inhibitors enhanced it. The possible relevance of this phenomenon in other systems is discussed.
Assuntos
Antígenos HLA-D/imunologia , Linfócitos T/parasitologia , Theileria parva/crescimento & desenvolvimento , Theileriose/imunologia , Animais , Cálcio/metabolismo , Ciclo Celular , Expressão Gênica , Genes myc , Humanos , Técnicas In Vitro , Interleucina-2/genética , Isoanticorpos/imunologia , Ativação Linfocitária , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , Receptores de Interleucina-2/genética , Linfócitos T/citologiaRESUMO
The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-kappa B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-kappa B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-kappa B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-kappa B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-kappa B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-kappa B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-kappa B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-kappa B. We propose that high levels of activated NF-kappa B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite.
Assuntos
Apicomplexa/fisiologia , Linfócitos T/parasitologia , Fatores de Transcrição/biossíntese , Animais , Bovinos , Linhagem Celular , Núcleo Celular/análise , Cloranfenicol O-Acetiltransferase/genética , Citoplasma/análise , Eletroforese em Gel de Poliacrilamida , Elementos Facilitadores Genéticos , Guanosina Trifosfato/farmacologia , HIV-1/genética , NF-kappa B , Plasmídeos , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Theileriose/parasitologia , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
The serine protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-kappaB activation in T. parva-transformed T-cells, with phosphorylation of IkappaBalpha and IkappaBbeta being inhibited with different kinetics. TPCK-mediated inhibition of IkappaB phosphorylation, NF-kappaB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kappaB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kappaB activation pathway(s).
Assuntos
Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Células Jurkat/citologia , Inibidores de Serina Proteinase/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Animais , Anexina A5/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/parasitologia , Cisteína/farmacologia , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Humanos , Proteínas I-kappa B , Células Jurkat/efeitos dos fármacos , Células Jurkat/parasitologia , NF-kappa B/metabolismo , Fosforilação , Theileria parva , Theileriose/imunologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Theileria annulata is a tick-transmitted protozoan parasite of cattle, which transforms cells of macrophage (Mphi) or B cell lineage. Bone marrow cells, bone marrow cell-derived, and monocyte-derived Mphi were infected with T. annulata sporozoites, and the resulting cell lines were assessed for surface marker expression and function. Transformed lines expressed histocompatibility complex (MHC) class-I and II, CD44, CD45, and the myeloid marker DH598-surface markers CD14, CD11b, M-M7, TH57A, and to a lesser extent CD11a/CD18, CD11c, and ACT(B), were down-regulated. Likewise, transformed cells failed to express Mphi functions (Fc-receptor-mediated phagocytosis, phorbol myristate acetate-induced oxidative burst, lipopolysaccharide-induced tumor necrosis factor alpha, and nitric oxide generation and procoagulant activity up-regulation). Mphi origin was assured by homogeneity of the starting population, cloning of cells by limiting dilution, and repeated microscopic and flow cytometric monitoring of the cell lines. Elimination of the parasite by treatment with BW720c resulted in the re-acquisition of monocyte lineage properties, as evidenced by up-regulation of CD14, and by re-acquisition of the capacity to ingest opsonized sheep red blood cells and bacteria. Thus, Mphi transformed by T. annulata appear to undergo a process of parasite-induced dedifferentiation but reassume the differentiated phenotype upon elimination of the parasite.
Assuntos
Macrófagos/fisiologia , Macrófagos/parasitologia , Theileria annulata , Animais , Antígenos de Superfície/análise , Antiprotozoários/farmacologia , Bovinos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Transformada , Regulação para Baixo , Feminino , Citometria de Fluxo , Interações Hospedeiro-Parasita , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Naftoquinonas/farmacologia , Fenótipo , Sensibilidade e Especificidade , Theileriose/tratamento farmacológico , Theileriose/patologiaRESUMO
In an electron microscopic investigation of the entry of sporozoites of Theileria parva into bovine lymphocytes, the fate of the surface coat of the parasite was traced by immunocytochemical methods. A monoclonal antibody (MAbD1) raised in mice and directed against a surface antigen of sporozoites, was applied to ultrathin frozen sections of bovine lymphocytes infected in vitro. Sites of binding of MAbD1 were localized using a protein A-colloidal gold conjugate as an electron-dense label. The surface of all free sporozoites was labelled. Sporozoites in the process of entering were labelled only on that portion of the membrane not yet tightly bound to the lymphocyte membrane. No label was detected on sporozoites that had completed entry. After fixation with formaldehyde, but not with glutaraldehyde, local areas of labelling were found on lymphocytes in contact with sporozoites and on cells already invaded. The sporozoite organelles, called micronemes, occasionally appeared to contain labelled antigen. No label was found on sporozoites or lymphocytes in control preparations previously exposed to non-specific antibody or treated with protein A-colloidal gold alone. The findings support the conclusion that the sporozoite surface coat, containing the antigen recognized by MAbD1, is shed as the sporozoite enters the host cell.
Assuntos
Apicomplexa/fisiologia , Linfócitos/parasitologia , Theileriose/parasitologia , Animais , Apicomplexa/imunologia , Apicomplexa/ultraestrutura , Bovinos , Membrana Celular/fisiologia , Técnicas In Vitro , Linfócitos/ultraestrutura , Microscopia Eletrônica , Theileriose/sangueRESUMO
A human interleukin 4 (hIL-4)-encoding cDNA (hIL4) probe was used to screen a bovine genomic library, and three clones containing sequences with homology to the human and mouse IL4 cDNAs were isolated. Sequence information obtained from one of these genomic clones was used to design an oligodeoxyribonucleotide primer corresponding to the transcription start point region for use in the polymerase chain reaction (PCR). The PCR-RACE protocol, designed for the rapid amplification of cDNA ends, was successfully used to generate a full-length bovine IL4 (bIL4) cDNA clone from polyadenylated RNA isolated from concanavalin A-stimulated bovine lymph node cells. The bIL4 cDNA is 570 bp in length and contains an open reading frame of 405 nucleotides (nt), coding for a 15.1-kDa precursor of 135 amino acids (aa), which should be reduced to 12.6 kDa for unglycosylated bIL4 after cleavage of a putative hydrophobic leader sequence of 24 aa. The aa sequence contains one possible Asn-linked glycosylation site. Bovine IL4 is shorter than mouse (mIL4) and hIL4, because of a 51-nt deletion in the coding region. Comparison of the overall nt and deduced aa sequences shows a greater homology of bIL4 with hIL4 than with mIL4. This homology is not evenly distributed, however, with the nt sequences 5' and 3' of the coding region showing a much greater homology between all three species than the coding sequence.
Assuntos
Clonagem Molecular/métodos , Interleucina-4/genética , Reação em Cadeia da Polimerase , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Bovinos , DNA , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Homologia de Sequência do Ácido NucleicoRESUMO
CD3 epsilon and the zeta-chain of the bovine T-cell receptor (TCR) are two invariant molecules with an important role in signal transduction via the TCR/CD3 complex. The nucleotide sequence of a bovine CD3 epsilon cDNA clone containing the complete coding sequence was determined and the deduced amino acid (aa) sequence compared to that of other species. The cytoplasmic domains of the different CD3 epsilon clearly show a higher degree of conservation than the extracellular domains. Bovine CD3 epsilon produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3 epsilon. A partial bovine TCR zeta-chain cDNA was generated by the polymerase chain reaction (PCR) using primers that were based on sequences that are conserved between different species; 3' and 5' RACE-PCR were carried out to obtain the complete TCR zeta-chain cDNA sequence. A comparison of the predicted TCR zeta-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCR zeta-chains. In contrast to CD3 epsilon, the short extracellular domain of the TCR zeta-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity. Ab were raised against the TCR zeta-chain, produced as a glutathione S-transferase fusion protein in E. coli, and were used in Western blot analysis to further characterise TCR zeta-chain expression in T-cells. The regents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells.
Assuntos
Complexo CD3/genética , Proteínas de Membrana/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Complexo CD3/química , Bovinos , Linhagem Celular , Clonagem Molecular , DNA Complementar , Escherichia coli , Expressão Gênica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/química , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/genética , Análise de Sequência , Linfócitos T/citologiaRESUMO
Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to kappa B-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells.
Assuntos
Ativação Linfocitária , NF-kappa B/metabolismo , Linfócitos T/metabolismo , Linfócitos T/parasitologia , Theileria parva/fisiologia , Fator 2 Ativador da Transcrição , Transporte Ativo do Núcleo Celular , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , NF-kappa B/genética , Subunidade p50 de NF-kappa B , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Fator de Transcrição RelA , Fator de Transcrição RelB , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
A hybridoma cell line has been previously produced which secretes monoclonal antibodies able to neutralize sporozoites of Theileria parva, the causative agent of East Coast fever of cattle. Cells from this line were injected intra-peritoneally into pristane-treated BALB/c mice. During the last 4 days of hybridoma cell growth, mice were given 4 daily intraperitoneal injections of a mixture of tritiated amino acids in order to biosynthetically radiolabel the monoclonal antibody being produced in ascites fluid. The specific activity of the antibody obtained was 100 mCi/mmol. The labelled antibody was used to detect, by autoradiography, a surface coat antigen of T. parva sporozoites in cryostat sections of Theileria-infected tick salivary glands. The method allows the preparation of large quantities of biosynthetically radiolabelled immunological probes for the detection of immunoreactive sites in biological specimens.
Assuntos
Anticorpos Monoclonais/biossíntese , Antígenos de Protozoários/análise , Apicomplexa/imunologia , Líquido Ascítico , Animais , Anticorpos Monoclonais/imunologia , Apicomplexa/análise , Hibridomas/imunologia , Hibridomas/transplante , Insetos Vetores/análise , Marcação por Isótopo/métodos , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Theileriose/imunologia , Carrapatos/análiseRESUMO
Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.
Assuntos
Endopeptidases/genética , Fasciola hepatica/genética , Genes de Helmintos , Proteínas de Helminto/genética , Família Multigênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Escherichia coli , Fasciola hepatica/crescimento & desenvolvimento , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Using a rapid procedure to isolate schizonts of the intracellular parasite Theileria parva from infected bovine lymphocytes, we have prepared parasite RNA that is more than 90% pure. Characterization of this schizont RNA has revealed the presence of two ribosomal RNA species of 3.3 kb and 1.8 kb and a third non-adenylated abundant RNA species of 1.9 kb. In vitro translation of the isolated schizont mRNA has identified about 200 parasite specific polypeptides, only a few of which could be detected by translation of mRNA from infected host cells. By analysing the kinetics of liquid hybridization of schizont mRNA with its homologous complementary DNA the nucleotide sequence complexity of the abundant class of the parasite mRNA has been estimated to be 1.7 x 10(3) kb. Assuming a number average size of 2 kb per mRNA molecule this would represent 4000 transcripts for all abundance classes of the schizont mRNA. Using the same technique we estimate that approximately 10% of the mRNA isolated from infected lymphocytes were transcripts from the parasite genome. We conclude that the low number of parasite specific translation products in the mRNA from infected lymphocytes and the low number of parasite proteins detected in isolated schizonts reported previously is due to the low abundance of the parasite transcripts rather than a low number of expressed parasite genes.
Assuntos
Apicomplexa/genética , Linfócitos/parasitologia , RNA Mensageiro/isolamento & purificação , RNA Ribossômico/isolamento & purificação , RNA/isolamento & purificação , Animais , Bovinos , Linhagem Celular , Reações Cruzadas , Eletroforese em Gel de Ágar , Eletroforese em Gel Bidimensional , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , RNA/análise , RNA Mensageiro/análise , RNA Ribossômico/análise , Theileriose/parasitologiaRESUMO
Sixteen monoclonal antibodies, raised against macroschizonts of Theileria parva, were tested against 10 different stocks of the parasite. The indirect fluorescent antibody test was used to demonstrate that these antibodies showed different binding affinities to macroschizonts of the various stocks. A profile of antibody binding could thus be prepared for each stock. For a given stock the profile was consistently the same irrespective of culture passage level, host cell background and method of antigen preparation. Monoclonal antibody profiles thus appear to provide a means of characterisation of stocks of T parva in vitro, and preliminary evidence suggests that profiles may be used to differentiate strains. The best source of antigen for testing theilerial stocks was macroschizont infected cells raised in culture, but suitable preparations could also be made from lymph node biopsies of cattle infected with East Coast fever. In a field outbreak of disease it might thus be possible rapidly to characterise the strains of T parva involved and plan immunisation and control measures accordingly.
Assuntos
Anticorpos Monoclonais/imunologia , Apicomplexa/imunologia , Theileriose/imunologia , Animais , Apicomplexa/classificação , Bovinos , Técnicas Imunológicas , QuêniaRESUMO
Stocks of Theileria parva, which had been characterised by monoclonal antibody profiles, were used to challenge cattle previously immunised against East Coast fever (ECF). When cattle were subjected to homologous challenge, or heterologous challenge with a stock of identical profile to that which had initiated immunity, they showed mild or inapparent reactions. However, when cattle were challenged with a stock of a different profile many underwent severe or fatal ECF reactions. Thus, there appears to be good correlation between cross resistance patterns in vivo and parasite differences detected in vitro by monoclonal antibodies. The results indicate that monoclonal antibody profiles can be used to characterise strains of T parva in vitro, and thus provide valuable data for planning field immunisation programmes. Now that monoclonal antibodies offer the potential of characterising theilerial parasites so precisely, the need arises for more disciplined use of terms describing parasite populations and collections. It is proposed that the rules of nomenclature devised for trypanosomes be adopted for Theileria species.
Assuntos
Anticorpos Monoclonais/imunologia , Apicomplexa/imunologia , Theileriose/imunologia , Vacinação , Animais , Apicomplexa/classificação , Bovinos , Reações Cruzadas , Técnicas Imunológicas , Quênia , Terminologia como Assunto , Theileriose/prevenção & controleRESUMO
The rapid population growth in subsaharan Africa necessitates a great increase in animal production in the more humid zones. Vector-borne diseases occurring in these zones will assume more importance, but are difficult to control. They include theileriosis and heartwater. Recent developments in research on these diseases are presented. Indigenous animal populations in endemic areas, subjected to natural selection, are far less susceptible than exotic stock. Heartwater, caused by the rickettsia Cowdria ruminantium, transmitted by Amblyomma ticks, causes high mortality in exotic ruminants. It has received much attention in recent years, partly because the disease has been introduced from Africa into the Caribbean and threatens the American mainland. Since the recent success of in vitro culture, much progress in research has been made, but so far prevention still relies mainly on acaricidal tick control; an infection and treatment method is used on a limited scale. Antigenic diversity is a complication for immunization procedures. Theileria parva (East Coast fever, Corridor disease and January disease) and T.annulata (Mediterranean or tropical theileriosis) are the most pathogenic of the 6 species of this protozoan genus that infect cattle. Great progress has been made in recent years in knowledge on the immunology, the epidemiology, the taxonomy and the chemotherapy of theileriosis. Intensive acaricidal tick control can now be supplemented by an attenuated schizont vaccine against T.annulata, while immunization against East Coast fever is carried out on a limited scale using virulent sporozoite infection and treatment. Research on recombinant vaccines is promising. Antigenic diversity in T.parva is a serious complication.
Assuntos
Ehrlichia ruminantium , Hidropericárdio , Theileria parva , Theileriose , Infestações por Carrapato/veterinária , África/epidemiologia , Animais , Vetores Aracnídeos , Bovinos , Hidropericárdio/epidemiologia , Pesquisa/tendências , Theileriose/epidemiologia , Theileriose/transmissão , Controle de Ácaros e Carrapatos/tendênciasRESUMO
The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.
Assuntos
Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Esquizontes/metabolismo , Theileria annulata/crescimento & desenvolvimento , Theileria annulata/metabolismo , Theileriose/parasitologia , Animais , Bovinos , Espectrometria de Massas , Dados de Sequência Molecular , Proteômica , Proteínas de Protozoários/genética , Esquizontes/química , Esquizontes/crescimento & desenvolvimento , Theileria annulata/química , Theileria annulata/genéticaRESUMO
The protozoan intracellular parasites, Theileria parva and Theileria annulata, infect cattle and cause severe and fatal leukocytic proliferative diseases. The proliferation is dependent on the presence of the parasites in the host cell cytoplasm. T. parva-infected cells proliferate permanently in cell culture and exhibit many features characteristic of tumor cells. The proliferation is reversible by treatment with parasite-specific drugs. Constitutive expression of interleukin-2, its receptor and their transcription factor, NF-kappa B, are dependent on the parasite and suggest autocrine growth. Cell-cell contact possibly via T cell adhesion molecules has been shown to stimulate proliferation.
Assuntos
Ativação Linfocitária , Linfócitos/parasitologia , Theileria parva/fisiologia , Animais , Bovinos , Comunicação Celular , Linhagem Celular Transformada , Citocinas/fisiologia , Citoplasma/parasitologia , Linfócitos/fisiologiaRESUMO
Ceramide is a lipid second messenger which is generated in response to stimulation of a number of surface receptors, treatment with chemotherapeutic agents, or ionising radiation. Depending on the target cell, ceramide induces diverse biological responses including apoptosis, cell-cycle arrest, differentiation, and also proliferation. We studied the effect of ceramide on the degradation of IkappaB, the cytoplasmic inhibitor of the transcription factor NF-kappaB. We show that ceramide treatment results in reduced levels of phosphorylated IkappaBalpha and degradation of both IkappaBalpha and IkappaBbeta. Ceramide synergised with okadaic acid (OA), a compound which interferes with the protein phosphatase 2A-controlled component of the NF-kappaB activation pathway, enhancing OA-induced IkappaB degradation. Ceramide also synergised with phorbol 12-myristate 13-acetate, which mimics protein kinase C activation. Finally, we show that the synergistic effect of ceramide with OA or phorbol ester can be observed in primary lymph node T-cells as well as in transformed T-cells.