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Sailen Barik

Publications and source records attributed to Sailen Barik.

27 records · Page 2Linked to original sources
PubMed2003

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 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.

Adenosine Triphosphate↗
PubMed2003

Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus.

BACKGROUND: Actin is required for the gene expression and morphogenesis of respiratory syncytial virus (RSV), a clinically important Pneumovirus of the Paramyxoviridae family. In HEp-2 cells, RSV infection also induces actin stress fibers, which may be important in the immunopathology of the RSV disease. Profilin, a major regulator of actin polymerization, stimulates viral transcription in vitro. Thus, we tested the role of profilin in RSV growth and RSV-actin interactions in cultured cells (ex vivo). RESULTS: We tested three cell lines: HEp-2 (human), A549 (human), and L2 (rat). In all three, RSV grew well and produced fused cells (syncytium), and two RSV proteins, namely, the phosphoprotein P and the nucleocapsid protein N, associated with profilin. In contrast, induction of actin stress fibers by RSV occurred in HEp-2 and L2 cells, but not in A549. Knockdown of profilin by RNA interference had a small effect on viral macromolecule synthesis but strongly inhibited maturation of progeny virions, cell fusion, and induction of stress fibers. CONCLUSIONS: Profilin plays a cardinal role in RSV-mediated cell fusion and viral maturation. In contrast, interaction of profilin with the viral transcriptional proteins P and N may only nominally activate viral RNA-dependent RNA polymerase. Stress fiber formation is a cell-specific response to infection, requiring profilin and perhaps other signaling molecules that are absent in certain cell lines. Stress fibers per se play no role in RSV replication in cell culture. Clearly, the cellular architecture controls multiple steps of host-RSV interaction, some of which are regulated by profilin.

Animals↗
PubMed2003

Characterization of a unique aspartate-rich protein of the SET/TAF-family in the human malaria parasite, Plasmodium falciparum, which inhibits protein phosphatase 2A.

A search for physiological inhibitors of protein phosphatases led to the identification of a Plasmodium falciparum (Pf) cDNA that had the potential to code for an aspartate-rich protein and hence named ARP. The PfARP was virtually identical to its Plasmodium berghei counterpart in gene structure and protein sequence. The PfARP coding sequence contained two introns, and the predicted protein contained 269 amino acid residues. Its primary structure showed significant similarity to eukaryotic proteins of the SET and TAF-family that included two inhibitors of mammalian serine/threonine protein phosphatase 2A (PP2A), namely I1(PP2A) and I2(PP2A). Like the SET and TAF proteins, it had an extremely acidic tail. The cDNA was confirmed by recombinant expression in bacteria. Native parasitic ARP was purified and was found to be highly thermostable. PfARP specifically inhibited the parasitic PP2A at nanomolar concentrations, with no effect on PP1, PP2B, PP5, or PPJ. Expression of PfARP in HeLa cells led to elevated phosphorylation of c-Jun, and activation of transcription factors AP1 and NF-kappa B. These functional properties are also characteristic of the SET/TAF-family proteins. The ARP mRNA and protein were detectable in all the erythrocytic asexual stages of the parasite, and the protein was located mainly in the parasitic cytoplasm. Thus, PfARP is a unique cytoplasmic member of the SET/TAF-family and a candidate physiological regulator of the Plasmodium PP2A.

Amino Acid Sequence↗
PubMed2003

Kinetic and site-directed mutagenesis studies of prevotella intermedia acid phosphatase.

Site-directed mutagenesis was used to examine the roles of the conserved histidine, arginine and cysteine residues in acid phosphatase from Prevotella intermedia (PiACP). The replacement of histidine and arginine residues resulted in the elimination of the PiACP activity while the cysteine mutants retained activity. These results suggest that the histidine and arginine residues are essential for catalysis.

Acid Phosphatase↗
PubMed2002

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.

Animals↗
PubMed2002

Megaprimer PCR.

Explore the source record for details and available documents.

DNA Primers↗
PubMedDate not supplied

New drugs and treatment for respiratory syncytial virus.

The respiratory syncytial virus (RSV) is a global health problem affecting infants and the elderly and claiming more lives than AIDS in many parts of the world. Only two antibody drugs are approved for its prevention, and ribavarin, a relatively nonspecific antiviral, is used for treatment. In the mid-1990s, a number of pharmaceutical and biotech companies initiated research programs against RSV. Together, the academic and the industrial R&D covered the whole spectrum of antibodies, vaccines, synthetic small molecule antiviral and antisense technology, and at one point, accounted for at least 25 active R&D programs. However, coincident to the marketing of the monoclonal antibody palivizumab (Synagis) in 1998, a sharp decline in such projects ensued. Many companies recently cancelled RSV projects during a prioritisation of their R&D portfolios although the continuing medical need, large market size and sales projections clearly indicate that a safe and effective RSV drug or vaccine is likely to attain blockbuster status. Today RSV receives an insignificant fraction of the R&D budget compared with AIDS, for example. This article reviews the present status of the anti-RSV regimen, covers drugs in the market and in development, and attempts to link basic research to industrial drug development, animal models of RSV, clinical trials, current clinical management, and present and future market projections. It is hoped that the unmet medical need of the victims of RSV will encourage continued involvement of the pharmaceutical and biotechnology industry in developing safe and effective prevention and treatments for RSV.

Aged↗