The heat shock protein 90 of Plasmodium falciparum and antimalarial activity of its inhibitor, geldanamycin

BACKGROUND: The naturally occurring benzoquinone ansamycin compound, geldanamycin (GA), is a specific inhibitor of heat shock protein 90 (Hsp90) and is a potential anticancer agent. Since Plasmodium falciparum has been reported to have an Hsp90 ortholog, we tested the possibility that GA might inhibit it and thereby display antiparasitic activity. RESULTS: We provide direct recombinant DNA evidence for the Hsp90 protein of Plasmodium falciparum, the causative agent of fatal malaria. While the mRNA of Hsp90 was mainly expressed in ring and trophozoite stages, the protein was found in all stages, although schizonts contained relatively lower amounts. In vitro the parasitic Hsp90 exhibited an ATP-binding activity that could be specifically inhibited by GA. Plasmodium growth in human erythrocyte culture was strongly inhibited by GA with an IC(50 )of 20 nM, compared to the IC(50 )of 15 nM for chloroquine (CQ) under identical conditions. When used in combination, the two drugs acted synergistically. GA was equally effective against CQ-sensitive and CQ-resistant strains (3D7 and W2, respectively) and on all erythrocytic stages of the parasite. CONCLUSIONS: Together, these results suggest that an active and essential Hsp90 chaperone cycle exists in Plasmodium and that the ansamycin antibiotics will be an important tool to dissect its role in the parasite. Additionally, the favorable pharmacology of GA, reported in human trials, makes it a promising antimalarial drug

Psychological and Pedagogical Means of Monitoring the Development of Listening Skills

Статтю присвячено проблемі розвитку навичок аудіювання в навчальному процесі з вивчення іноземної мови. Автори діляться досвідом використання допоміжних психолого-методичних засобів; серед них тести, опитувальники, шкали діагностики та самодіагностики. Надано докладні пояснення щодо конструкції, можливих методичних прийомів та організаційних аспектів «Шкали діагностики та самодіагностики рівня аудіювання іншомовного тексту». Шкала самооцінки сприйняття і розуміння іншомовного тексту забезпечує викладача об’єктивними відомостями про рівень розвитку цієї навички в групи загалом та в кожного студента зокрема і є засобом залучення в процес слухання, допомагає правильно диференціювати й оцінювати навички та вміння членів групи, що, у свою чергу, стимулює розроблення і використання різноманітних методів і прийомів навчання аудіювання, тобто до індивідуалізації процесу навчання в умовах спільної групової навчально-пізнавальної діяльності, а отже, досягнення спільно-розподіленої навчальної ефективності. Застосування опитувальників на зразок «Ваше вміння слухати», а також тесту «Ступінь комунікативного контролю» англійською мовою під час цілеспрямованого навчання аудіюванню на заняттях з іноземної мови сприяють удосконаленню навичок контролю за власним мовленням і мовленнєвою діяльністю партнера. Автори пропонують низку методів та прийомів, які були апробовані в їхніх вишах на заняттях з англійської мови, та продемонстрували позитивний результат. The article deals with the problem of the development of listening skills at the lessons of foreign languages. A range of methods and techniques is proposed. The authors share their experience in the sphere of usage the supplementary methods such as questionnaires, scales of diagnostics and selfdiagnostics, psychological tests, etc. “Diagnostic and self-diagnostic scale of listening skills”, its structure and different ways of teaching techniques are given. This scale provides a teacher with the objective data of listening skill development of each student and group as a whole. This method helps to involve each student into the listening task, stimulates an adequate differentiation and assessment of one’s listening skills. In its turn it stimulates the development and implementation of different teaching techniques. It leads to individual and group effectiveness. Such questionnaires as “Are you a good listener?” and “Level of your communicative control” may be a good stimulus for conscious speaking control. These techniques were used for foreign language teaching and proved to be efficient

Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

BACKGROUND: Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. RESULTS: We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA) led to inhibition of parasite DNA synthesis. CONCLUSIONS: The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes

Post-translational generation of constitutively active cores from larger phosphatases in the malaria parasite, Plasmodium falciparum: implications for proteomics

BACKGROUND: Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum. RESULTS: P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca(+2)-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca(+2). We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca(+2), the processed cores are constitutively active and either less responsive or unresponsive to Ca(+2). The processing is extremely rapid, specific, and occurs in vivo. CONCLUSIONS: Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation

The heat shock protein 90 of <it>Plasmodium falciparum </it>and antimalarial activity of its inhibitor, geldanamycin

Abstract Background The naturally occurring benzoquinone ansamycin compound, geldanamycin (GA), is a specific inhibitor of heat shock protein 90 (Hsp90) and is a potential anticancer agent. Since Plasmodium falciparum has been reported to have an Hsp90 ortholog, we tested the possibility that GA might inhibit it and thereby display antiparasitic activity. Results We provide direct recombinant DNA evidence for the Hsp90 protein of Plasmodium falciparum, the causative agent of fatal malaria. While the mRNA of Hsp90 was mainly expressed in ring and trophozoite stages, the protein was found in all stages, although schizonts contained relatively lower amounts. In vitro the parasitic Hsp90 exhibited an ATP-binding activity that could be specifically inhibited by GA. Plasmodium growth in human erythrocyte culture was strongly inhibited by GA with an IC50 of 20 nM, compared to the IC50 of 15 nM for chloroquine (CQ) under identical conditions. When used in combination, the two drugs acted synergistically. GA was equally effective against CQ-sensitive and CQ-resistant strains (3D7 and W2, respectively) and on all erythrocytic stages of the parasite. Conclusions Together, these results suggest that an active and essential Hsp90 chaperone cycle exists in Plasmodium and that the ansamycin antibiotics will be an important tool to dissect its role in the parasite. Additionally, the favorable pharmacology of GA, reported in human trials, makes it a promising antimalarial drug.</p

Viral Infection of the Lungs through the Eye

Respiratory syncytial virus (RSV) is the foremost respiratory pathogen in newborns and claims millions of lives annually. However, there has been no methodical study of the pathway(s) of entry of RSV or its interaction with nonrespiratory tissues. We and others have recently established a significant association between allergic conjunctivitis and the presence of RSV in the eye. Here we adopt a BALB/c mouse model and demonstrate that when instilled in the live murine eye, RSV not only replicated robustly in the eye but also migrated to the lung and produced a respiratory disease that is indistinguishable from the standard, nasally acquired RSV disease. Ocularly applied synthetic anti-RSV small interfering RNA prevented infection of the eye as well as the lung. RSV infection of the eye activated a plethora of ocular cytokines and chemokines with profound relevance to inflammation of the eye. Anticytokine treatments in the eye reduced ocular inflammation but had no effect on viral growth in both eye and lung, demonstrating a role of the cytokine response in ocular pathology. These results establish the eye as a major gateway of respiratory infection and a respiratory virus as a bona fide eye pathogen, thus offering novel intervention and treatment options

Respiratory Syncytial Virus Nonstructural Proteins Decrease Levels of Multiple Members of the Cellular Interferon Pathways▿

Viruses of the Paramyxoviridae family, such as the respiratory syncytial virus (RSV), suppress cellular innate immunity represented by type I interferon (IFN) for optimal growth in their hosts. The two unique nonstructural (NS) proteins, NS1 and NS2, of RSV suppress IFN synthesis, as well as IFN function, but their exact targets are still uncharacterized. Here, we investigate if either or both of the NS proteins affect the steady-state levels of key members of the IFN pathway. We found that both NS1 and NS2 decreased the levels of TRAF3, a strategic integrator of multiple IFN-inducing signals, although NS1 was more efficient. Only NS1 reduced IKKɛ, a key protein kinase that specifically phosphorylates and activates IFN regulatory factor 3. Loss of the TRAF3 and IKKɛ proteins appeared to involve a nonproteasomal mechanism. Interestingly, NS2 modestly increased IKKɛ levels. In the IFN response pathway, NS2 decreased the levels of STAT2, the essential transcription factor for IFN-inducible antiviral genes. Preliminary mapping revealed that the C-terminal 10 residues of NS1 were essential for reducing IKKɛ levels and the C-terminal 10 residues of NS2 were essential for increasing and reducing IKKɛ and STAT2, respectively. In contrast, deletion of up to 20 residues of the C termini of NS1 and NS2 did not diminish their TRAF3-reducing activity. Coimmunoprecipitation studies revealed that NS1 and NS2 form a heterodimer. Clearly, the NS proteins of RSV, working individually and together, regulate key signaling molecules of both the IFN activation and response pathways

Mutational Analysis Reveals a Noncontractile but Interactive Role of Actin and Profilin in Viral RNA-Dependent RNA Synthesis▿

As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role