Ribonuclease-resistant RNA controls and standards.
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RNA↗
Biomedical subjects
B L Pasloske
Publications and source records attributed to B L Pasloske.
Ribonuclease-resistant RNA controls (Armored RNA) for reverse transcription-PCR, branched DNA, and genotyping assays for hepatitis C virus.
BACKGROUND: Comparison and evaluation of molecular diagnostic assays for the detection and quantification of hepatitis C virus (HCV) RNA have been limited by the lack of RNA controls and calibrators. Armored RNA technology is a means for producing RNA that is completely protected from plasma ribonucleases. This method produces recombinant pseudoviral particles that are noninfectious and contain predefined RNA sequences. METHODS: A consensus 412-base sequence from the 5'NCR/Core region of HCV subtype 2b was derived from 34 individually sequenced HCV genotype 2b variants. A DNA fragment encoding the consensus HCV-2b sequence was synthesized de novo, cloned, and expressed as an Armored RNA control. The resulting HCV-2b Armored RNA (AR-HCV-2b) contained the complete HCV-2b consensus RNA sequence encapsidated within a protective protein coat. RESULTS: AR-HCV-2b was fully recoverable from human plasma incubated at 4 degrees C for >300 days. The particles were tested in three clinical assay formats: Amplicor HCV Monitor 1.0, Quantiplex HCV RNA 2.0, and INNO-LiPA HCV II. When added into seronegative, nonviremic plasma, AR-HCV-2b showed reproducible signals and linear dilutions in both the Amplicor and Quantiplex assays. AR-HCV-2b was correctly identified as subtype 2b in the INNO-LiPA line probe assay. CONCLUSION: The HCV-2b Armored RNA control is a versatile, durable, ribonuclease-resistant viral RNA control that is compatible in three different clinical assay formats.
Base Sequence↗
Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards.
The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. "Armored RNA" is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at -20 degreesC or for 60 days at 4 degreesC. Additionally, Armored RNA preparations are homogeneous and noninfectious.
HIV-1↗
Identification of a region of PfEMP1 that mediates adherence of Plasmodium falciparum infected erythrocytes to CD36: conserved function with variant sequence.
Adherence of mature parasitized erythrocytes (PE) of Plasmodium falciparum to microvascular endothelial cells contributes directly to the virulence and pathology of this human malaria. The malarial variant antigen, P falciparum erythrocyte membrane protein 1 (PfEMP1), has been implicated as the PE receptor for CD36 on endothelial cells. We identified the region of PfEMP1 that mediates adherence of PE to CD36 and showed that a recombinant protein fragment from this region blocked and reversed adherence of antigenically different parasites. Sequence variation was evident in the CD36 binding domain of different PfEMP1 genes, yet many highly conserved residues, particularly cysteine residues, are evident. This suggests a highly conserved shape that mediates adherence to CD36. Immunization with the CD36-binding domain elicited sera that are cross-reactive with the different recombinant proteins but are strain-specific for the PE surface. Novel anti-adherence therapeutics and a malaria vaccine may derived from exploitation of the structure of the CD36 binding domain of PfEMP1.
Amino Acid Sequence↗
Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.
Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.
Animals↗
Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes.
Plasmodium falciparum-infected human erythrocytes evade host immunity by expression of a cell-surface variant antigen and receptors for adherence to endothelial cells. These properties have been ascribed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), an antigenically diverse malarial protein of 200-350 kDa on the surface of parasitized erythrocytes (PEs). We describe the cloning of two related PfEMP1 genes from the Malayan Camp (MC) parasite strain. Antibodies generated against recombinant protein fragments of the genes were specific for MC strain PfEMP1 protein. These antibodies reacted only with the surface of MC strain PEs and blocked adherence of these cells to CD36 but without effect on adherence to thrombospondin. Multiple forms of the PfEMP1 gene are apparent in MC parasites. The molecular basis for antigenic variation in malaria and adherence of infected erythrocytes to host cells can now be pursued.
Amino Acid Sequence↗
pTRIPLEscript: a novel cloning vector for generating in vitro transcripts from tandem promoters for SP6, T7 and T3 RNA polymerase.
We have constructed a family of novel in vitro transcription vectors in which functional T3, T7 and SP6 RNA polymerase promoters are arranged in tandem and directed towards a multiple cloning site. This prototype vector, named pTRIPLEscript, permits the transcription of one strand of a DNA insert by any of the three commonly used bacteriophage RNA polymerases with no apparent cross talk, i.e., use of the wrong promoter sequence. The vector has two main uses: (i) to clone probe sequences that will be distributed to many laboratories, allowing the use of the most convenient RNA polymerase; and (ii) to circumvent the problem of RNA polymerase-dependent premature termination.
Base Sequence↗
PfEMP3 and HRP1: co-expressed genes localized to chromosome 2 of Plasmodium falciparum.
A malarial protein, Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3), has been recently characterized as a high-molecular-mass component (approx. 315 kDa) localized to the erythrocyte membrane of knob-bearing (K+), cytoadherent (C+) mature stages of P. falciparum-parasitized erythrocytes (PE) [Pasloske et al., Mol. Biochem. Parasitol. 59 (1993) 59-72]. Knobless (K-), non-cytoadherent (C-) parasites of the same strain were shown to lack the PfEMP3 gene. In view of the biological importance of the knobby and cytoadherent phenotypes with regard to parasite virulence, we extended the analysis of PfEMP3 and its gene product to other K+/K- and C+/C- parasites. Previously, other studies have shown that the malarial protein, knob-associated histidine-rich protein 1 (HRP1), is also strongly correlated with knob expression. Here, we show that PfEMP3 and HRP1 were absent from all the K- parasites tested, including the Palo Alto (PA) K-C+ strain. This result demonstrates that PfEMP3 and HRP1 are not essential for cytoadherence. PfEMP3 was localized to chromosome 2 of the K+ parasites, within no more than 130 kb of HRP1, between the telomere and HRP1. Stage-specific analysis of the mRNA for HRP1 and PfEMP3 indicated maximal transcription of the genes in ring-stage parasites, with little or no mRNA present during the mature parasite stages. Analysis of PfEMP3 and HRP1 by immunofluorescence assay (IFA) revealed identical staining patterns of fixed PE at all stages of the asexual life cycle. Hence, PfEMP3 and HRP1 are adjacent to each other in chromosome 2, co-expressed temporally and their gene products co-localized to the PE membrane.
Animals↗
Malaria, the red cell, and the endothelium.
Erythrocytes infected with mature stages of Plasmodium falciparum malaria adhere to vascular endothelial cells in postcapillary venules of several organs. In some patients, infected cells also form rosettes with uninfected erythrocytes. The special pathology of acute cerebral malaria appears to result from excessive adherence of infected cells in cerebral vessels coupled with occlusion of cerebral blood flow in microvessels by infected cell rosettes. Several endothelial cell proteins have been identified as potential receptors for infected erythrocyte adherence to vascular endothelium, including thrombospondin, CD36, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1). The receptor on infected erythrocytes that mediates adhesion to endothelial cells has been identified as a very large malarial protein on infected cells called PfEMP1. PfEMP1 has been shown to bind to CD36 and thrombospondin in vitro. Antibody-mediated blockade or reversal of infected erythrocyte adherence to vascular endothelium is postulated not only to decrease the pathology of blood-stage malaria, but also to lead to infected cell destruction and clearance, especially in the spleen. PfEMP1 is therefore a prime candidate malarial protein for inclusion in a multicomponent asexual malaria vaccine.
Cell Adhesion Molecules↗
The promise of asexual malaria vaccine development.
This review presents several reasons to support the belief that an asexual vaccine for human malaria caused by Plasmodium falciparum will be developed. It lists many of the current vaccine candidates that we think are ready for human trials and it summarizes a strategy for the development of an asexual vaccine.
Animals↗
Cloning and characterization of a Plasmodium falciparum gene encoding a novel high-molecular weight host membrane-associated protein, PfEMP3.
The rat monoclonal antibody, mAb 12C11, reacts with numerous proteins from mature asexual stages of Plasmodium falciparum. The largest is 315 kDa and is designated PfEMP3. A lambda gt11 expression library, generated from genomic DNA of Malayan Camp strain parasites, was screened with mAb 12C11. One positive clone, lambda 12.1.3, contained a 1.4-kb fragment in frame with the beta-galactosidase gene of lambda gt11. The deduced 455-amino acid sequence is a novel, highly charged sequence encoding two 15-amino acid repeats at the N-terminus followed by 27 repeats of 13 amino acids. The last 59 C-terminal residues are non-repetitive. Two in-frame stop codons at the 3' end of the DNA suggests that this DNA fragment encodes the C-terminus of the protein. Southern blotting with the cloned fragment identified two copies of this fragment per haploid genome in knob-positive, parasitized erythrocytes (K+PE). Both DNA fragments are absent from K - PE. Northern blotting of trophozoite-stage PE total RNA revealed mRNAs of 10, 4.4 and 2 kb in K+PE, but no hybridization with K - PE. Immune sera were elicited against the lambda 12.1.3 beta-galactosidase fusion protein and peptides generated from the predicted lambda 12.1.3 amino acid sequence. These sera and mAb 12C11 reacted specifically with PfEMP3 in Western blots of mature K+PE but not with K - PE. Rat and mouse sera against the recombinant protein produced an immunofluorescence pattern in fixed mature K+PE almost identical to the pattern produced by a monoclonal antibody against the knob-associated protein, Histidine Rich Protein 1. The same antibodies were immunofluorescence negative with fixed K - PE. Mouse antibodies against the recombinant protein reacted on immunoelectron microscopy with the erythrocyte membrane of K+PE, labeling knobs as well as the membrane between knobs. In contrast, a mAb against Histidine Rich Protein 1 reacted only under the electron dense material of knobs. We conclude that the lambda 12.1.3 clone encodes the C-terminal portion of the 315 kD PfEMP3 antigen and that PfEMP3 may be involved in knob formation or other perturbations of the erythrocyte membrane.
Amino Acid Sequence↗
Target antigens for asexual malaria vaccine development.
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Journal Article↗
Mutations in the fifth-position glutamate in Pseudomonas aeruginosa pilin affect the transmethylation of the N-terminal phenylalanine.
The pili of Pseudomonas aeruginosa are composed of 15-kDa pilin monomers that are synthesized in the cytoplasm and assembled in the membrane. Processing occurs between the synthesis and assembly steps. The propilin is cleaved by a unique leader peptidase encoded by pilD, which is adjacent to the pilin structural gene pilA. This generates an N-terminal phenylalanine that is subsequently methylated by an as yet uncharacterized transmethylase. The pili of P. aeruginosa belong to the type IV class of pilins, which share a highly conserved N-terminal region 35 amino acids in length, containing a short leader of 6 or 7 amino acids. Two site-specific mutants in the N-terminal region of the mature pilin were constructed. Reestablishing the fifth-position glutamate in a four amino acid deletion mutant (amino acids 4-7) restored the leader peptidase cleavage but not the methylation. A mutation of the fifth-position glutamate to alanine decreased the degree of methylation of the N-terminal phenylalanine. Pili were not assembled by these mutants as assessed by electron microscopy and sensitivity to pilus-specific bacteriophage. Methylation may be required for recognition of the pilin by the assembly machinery and is not residue specific. The fifth-position glutamate appears to play an important role in transmethylase recognition of the pilin subunit.
Amino Acid Sequence↗
An improved microassay for Plasmodium falciparum cytoadherence using stable transformants of Chinese hamster ovary cells expressing CD36 or intercellular adhesion molecule-1.
Stable transformants of Chinese hamster ovary (CHO) cell lines expressing high levels of human CD36 or intercellular adhesion molecule-1 (ICAM-1) have been produced as target cells for cytoadherence of Plasmodium falciparum-infected erythrocytes. An improved adherence microassay has been designed using small sample volumes and allowing convenient and reliable measurements on a large number of samples. The assay can be used both with purified proteins spotted on plastic and with the stably transformed CHO cell lines. The same assay plate can be evaluated either microscopically or by scintillation counting after use of 3H-hypoxanthine-labeled parasites. Using the microassay, functional expression of the transfected receptor molecules on CHO-CD36 and CHO-ICAM was confirmed using parasites with different cytoadherence phenotypes and cytoadherence inhibition experiments with a panel of anti-CD36 antibodies. The use of isolates from The Gambia confirmed the applicability of these assays for laboratory studies of these isolates.
Amino Acid Sequence↗
Immunochemical characterization and differentiation of two approximately 300-kD erythrocyte membrane-associated proteins of Plasmodium falciparum, PfEMP1 and PfEMP3.
Erythrocyte membrane-associated antigens of Plasmodium falciparum have been of long-standing interest as potential adherence receptors and vaccine candidates. We recently identified in trophozoite-stage infected erythrocytes a novel high molecular weight erythrocyte membrane-associated protein of P. falciparum, PfEMP3, defined by Western blotting with the rat monoclonal antibody 12C11. Genomic clone lambda 12.1.3 and cDNA clone p12.2 contain nucleic acid sequences encoding PfEMP3. Analysis of Malayan Camp strain parasites by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5% gels revealed that PfEMP3, defined by Western blot, has the same relative molecular weight (M(r)) as the surface-exposed protein PfEMP1 defined by cell surface iodination. We show here that PfEMP3 is distinct from PfEMP1 by three criteria. First, 125I-labeled PfEMP1 was resolved from PfEMP3 by extended migration on 4% gels. Second, in two strains of P. falciparum in which 125I-PfEMP1 has a different M(r), PfEMP3 had the same M(r). Third, immunization studies were performed with fusion proteins derived from clones lambda 12.1.3 and p12.2. Although one rabbit, Rb 05.75, immunized with the PfEMP3-derived fusion protein beta gal12.1.3, produced a serum that strongly immunoprecipitated PfEMP1 as well as PfEMP3, most sera immunoprecipitated only PfEMP3. Furthermore, immunoprecipitation of PfEMP3 by Rb 05.75 serum was blocked by the glutathione S-transferase 12.1.3 fusion protein, whereas immunoprecipitation of PfEMP1 was unaffected. Therefore, we conclude that PfEMP1 and PfEMP3 are antigenically distinct.
Amino Acid Sequence↗
The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum.
We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of greater than 200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, kappa), the other by clone 12C11 (IgM, lambda). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.
Animals↗
The role of polar pili in the adherence of Pseudomonas aeruginosa to injured canine tracheal cells: a semiquantitative morphologic study.
Pseudomonas aeruginosa adheres to respiratory epithelial cells in a highly specific fashion. In order to study the role of P. aeruginosa polar pili in the adherence process we conducted a quantitative morphological electron microscopic examination of P. aeruginosa adherence to SO2 injured canine tracheal cells in vitro. A pilin lacking background strain of P. aeruginosa PAK (BLP2) was constructed using a gene replacement and it in turn was engineered to express either the pilin gene of P. aeruginosa PAO, PAK, or no pilin gene. After 30 minutes incubation of these bacterial strains with injured canine tracheal rings the P. aeruginosa strains expressing pili adhered quantitatively more to the injured tracheal cells than did the pili lacking strains. PAO bearing strains adhered in greater numbers than PAK bearing strains. Healthy tracheal cells did not have any bacteria bound to their surfaces. The bacteria bound to the cilia and lateral edge of the exfoliating tracheal cells. Invasion of tracheal cells by piliated P. aeruginosa bacteria and penetration into the submucosa was also demonstrated. These data confirm the role of pili as important adhesins to injured tracheal cells. The difference in the adherence characteristics of pilin types PAK versus PAO may relate to the differences in the primary structure of these two pilin molecules.
Animals↗