| 1. |
- Ahuja, Sanjay, et al.
(författare)
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Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein I domains in Escherichia coli
- 2006
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Ingår i: Malaria Journal. - : Springer Science and Business Media LLC. - 1475-2875. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL ( Duffy binding like), CIDR ( cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally. Methods: A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli. Results: Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1 alpha (46.8%) > CIDR2 beta (42.9%) > DBL2-4 gamma (31.7%) > DBL2 beta + C2 (30.6%) > DBL1 alpha (24.9%) > DBL2-7 epsilon (23.1%) > DBL2-5 delta (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions. Conclusion: The acidic terminal segment ( ATS) and CIDR alpha/beta domain types are suitable for recombinant expression in E. coli while all DBL subtypes (alpha, beta, gamma, delta, epsilon) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains.
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| 2. |
- Albrecht, Letusa, et al.
(författare)
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var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2
- 2011
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Ingår i: Malaria Journal. - : BioMed Central. - 1475-2875 .- 1475-2875. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: The pathogenicity of Plasmodium falciparum is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra- vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of approximate to 60 var genes. Methods: The study of var gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorecence. Results: Transcripts from var1 (FCR3S1.2(var1); IT4var21) and other var genes were detected by semi-quantitative PCR but results from qPCR showed that one var gene transcript dominated over the others (FCR3S1.2var2; IT4var60). Antibodies raised in rats to the recombinant NTS-DBL1a of var2 produced in E. coli completely and dosedependently disrupted rosettes (approximate to 95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2var2 encodes the dominant PfEMP1 expressed in this parasite. Conclusion: The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2var2 (IT4var60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1a of FCR3S1.2var1 is likely due to cross-reactivity with NTS-DBL1 alpha of the var2 encoded PfEMP1.
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| 3. |
- Andersson, Annika, et al.
(författare)
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Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite
- 2020
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 287:13, s. 2744-2762
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Tidskriftsartikel (refereegranskat)abstract
- The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.
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| 4. |
- Aydin-Schmidt, Berit, et al.
(författare)
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Carolus Linnaeus, the ash, worm-wood and other anti-malarial plants
- 2010
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Ingår i: Scandinavian Journal of Infectious Diseases. - : Informa UK Limited. - 0036-5548 .- 1651-1980. ; 42:11-12, s. 941-942
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Tidskriftsartikel (refereegranskat)abstract
- In 1735 Carolus Linnaeus wrote that quinine was the preferred treatment for malaria but that the bark of the ash (Fraxinus excelsior) and worm-wood (Artemisia absinthium) also had effects on the disease. We here report that lipo- and hydrophilic extracts of the bark of the ash inhibit the in vitro growth of the asexual stages of P. falciparum. The data suggests that the knowledge of the treatment of malaria was already available in Europe some 300 years ago.
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| 5. |
- Bachmann, Julie, et al.
(författare)
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Affinity Proteomics Reveals Elevated Muscle Proteins in Plasma of Children with Cerebral Malaria
- 2014
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 10:4, s. e1004038-
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Tidskriftsartikel (refereegranskat)abstract
- Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.
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| 6. |
- Barragan, Antonio, et al.
(författare)
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Erythrocyte Glycans as Plasmodium falciparum Rosetting Receptors : Molecular Background of Strain Specific Rosette Disruption by Glycosaminoglycans and Sulfated Glycoconjugates
- 1999
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Ingår i: Experimental parasitology. - : Elsevier BV. - 0014-4894 .- 1090-2449. ; 91:2, s. 133-143
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Tidskriftsartikel (refereegranskat)abstract
- Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, is a virulent parasite phenotype associated with the occurrence of severe malaria, e.g., cerebral malaria. Compounds with specific anti-rosetting activity are potential therapeutic agents. Glycosaminoglycans and sulfated glycoconjugates were found to disrupt rosettes in a strain- and isolate-specific manner. Rosette disruption was strongly connected to the presence of N-sulfate groups in heparin/heparan sulfate as demonstrated by modified heparin preparations. This finding was corroborated by the disruption of rosettes with mono- and disaccharides derived from heparin/heparan sulfate that contained N-sulfated glucosamine. Furthermore, heparinase III treatment of erythrocyte cultures infected by FCR3S1 (and to some extent TM 284) P. falciparum strains abolished rosetting. Heparinase III treatment of the uninfected erythrocytes prior to mixing with the infected culture impeded formation of rosettes, indicating that the rosetting receptors at least partially are of glycosaminoglycan nature.
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| 7. |
- Brolin, M, et al.
(författare)
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Simultaneous transcription of duplicated var2csa gene copies in individual Plasmodium falciparum parasites
- 2009
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Ingår i: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 10:10, s. R117-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Single nucleotide polymorphisms are common in duplicated genes, causing functional preservation, alteration or silencing. The Plasmodium falciparum genes var2csa and Pf332 are duplicated in the haploid genome of the HB3 parasite line. Whereas the molecular function of Pf332 remains to be elucidated, VAR2CSA is known to be the main adhesin in placental parasite sequestration. Sequence variations introduced upon duplication of these genes provide discriminative possibilities to analyze allele-specific transcription with a bearing towards understanding gene dosage impact on parasite biology. Results: We demonstrate an approach combining real-time PCR allelic discrimination and discriminative RNA-FISH to distinguish between highly similar gene copies in P. falciparum parasites. The duplicated var2csa variants are simultaneously transcribed, both on a population level and intriguingly also in individual cells, with nuclear co-localization of the active genes and corresponding transcripts. This indicates transcriptional functionality of duplicated genes, challenges the dogma of mutually exclusive var gene transcription and suggests mechanisms behind antigenic variation, at least in respect to the duplicated and highly similar var2csa genes. Conclusions: Allelic discrimination assays have traditionally been applied to study zygosity in diploid genomes. The assays presented here are instead successfully applied to the identification and evaluation of transcriptional activity of duplicated genes in the haploid genome of the P. falciparum parasite. Allelic discrimination and gene or transcript localization by FISH not only provide insights into transcriptional regulation of genes such as the virulence associated var genes, but also suggest that this sensitive and precise approach could be used for further investigation of genome dynamics and gene regulation.
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| 8. |
- Chan, Sherwin, et al.
(författare)
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Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor
- 2017
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Ingår i: Nature Microbiology. - : Springer Science and Business Media LLC. - 2058-5276. ; 2:7
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Tidskriftsartikel (refereegranskat)abstract
- Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.
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| 9. |
- Ch'ng, Jun-Hong, et al.
(författare)
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Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.
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| 10. |
- Elfstrand, Lidia, et al.
(författare)
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From starch to starch microspheres: Factors controlling the microspheres quality
- 2006
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Ingår i: Stärke. - : Wiley. - 0038-9056. ; 58:8, s. 381-390
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Tidskriftsartikel (refereegranskat)abstract
- Starch microparticle is a dosage form suitable for encapsulation of protein drugs. The microparticle characteristics can be influential factors for consecutive sustained drug release from the starch matrix and impact its ability to be coated by poly(D,L-lactide-co-glycolide). In this study, six types of starch microparticles were investigated and characterized. Desired qualities of the microspheres such as spherical form of the particles, well-defined and sharp particle contours were coincident with coarse surface morphology, sharp and distinct peaks in DSC and X-ray graphs. It was concluded that the quality of the starch microparticles was influenced by molecular properties of the starch material as well as type of buffer, and these factors were related to recrystallization kinetics, amount and quality of ordered/crystalline structure of final starch microspheres and their microscopic appearance.
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