Platelet-derived growth factor production by cells from Dacron grafts implanted in a canine model.

PURPOSE: Previous studies of grafts implanted in dogs documented a time-dependent increase in platelet-derived growth factor (PDGF) production that correlated with inner-capsule thickness. The purpose of this study was to identify the cells in vascular grafts that produce PDGF. METHODS: Dacron thoracoabdominal grafts were seeded with autologous endothelial cells (ECs), implanted in 11 beagles, and removed after 4 or 20 weeks. ECs and smooth muscle cells (SMCs) were cultured from grafts and adjacent aorta, and PDGF in the conditioned media was measured by radioreceptor assay. The PDGF A-chain mRNA level in freshly harvested cells was assessed using reverse transcriptase, followed by polymerase chain reaction, and expressed as a ratio of glyceraldehyde-3-phosphate dehydrogenase signal. Localization of PDGF A-chain and B-chain protein was also examined with immunohistochemical analysis. RESULTS: Graft and aortic ECs in primary culture did not produce significantly different amounts of PDGF in 72 hours, averaging 368 +/- 160 and 340 +/- 81 pg/microgram DNA, respectively. Graft SMCs in primary culture produced significantly more PDGF than aortic SMCs (584 +/- 343 and 113 +/- 94 pg/microgram DNA, respectively; p < 0.01). Graft SMC PDGF secretion remained greater than aortic SMC PDGF secretion through at least six cell passages. PDGF A-chain mRNA levels were not significantly different for aortic or graft ECs. The PDGF A-chain mRNA level was significantly higher for graft SMCs than aortic SMCs (2.44 +/- 0.67 and 1.45 +/- 0.57 pg/microgram, respectively; p < 0.03). Immunocytochemical analysis detected PDGF A-chain and B-chain protein in the ECs from both native aorta and graft as well as the subendothelial SMCs in the graft, but not in the SMCs of the native aorta. CONCLUSIONS: These results suggest that graft SMCs are functionally altered, producing more PDGF than aortic SMCs. PDGF produced by graft SMCs may contribute to the development of intimal hyperplasia.

The gene encoding topoisomerase II from Plasmodium falciparum.

The gene for topoisomerase II has been isolated from genomic libraries of strain K1 of the human malarial parasite, Plasmodium falciparum. The sequence reveals an open reading frame of 4194 nucleotides which predicts a polypeptide of 1398 amino acids. There are apparently no introns. The sequence is present as a single copy which has an identity of 47.4% and a similarity of 65.4% with its human homologue. Sequences conserved in topoisomerase II from other species are present in Pftopoisomerase II but in addition it has two adjacent asparagine-rich insertions which are unique to it. We have also detected asparagine-rich regions in the gene for PfDNA polymerase alpha. The gene for Pftopoisomerase II has been localised to chromosome 14 and northern analysis reveals a transcript of 5.8 kb. Two independent antisera raised in mice against glutathione-S-transferase fusion proteins containing the amino terminal portion of the malarial protein detect a weak band on western blots at about 160kDa, the expected size of the protein. Use of the same antisera for immunofluorescence analysis suggests that the protein is present at all stages of intraerythrocytic growth of the parasite.

The gene encoding DNA polymerase alpha from Plasmodium falciparum.

The gene encoding DNA polymerase alpha from the human malaria parasite Plasmodium falciparum has been sequenced and characterised. The deduced amino acid sequence possesses the seven sequence motifs which characterise eukaryotic replicative DNA polymerases (I-VII) and four of five motifs (A-E) identified in alpha DNA polymerases. The predicted protein also contains sequences which are reminiscent of Plasmodium proteins but absent from other DNA polymerases. These include four blocks of additional amino acids interspersed with the conserved motifs of the DNA polymerases, four asparagine rich sequences and a novel carboxy-terminal extension. Repetitive sequences similar to those found in other malarial proteins are also present. cDNA-directed PCR was used to establish the presence of these features in the approximately 7kb mRNA. The coding sequence contains a single intron. The gene for DNAPol alpha is located on chromosome 4 and is transcribed in both asexual and sexual erythrocytic stages of the parasite.

Molecular characterisation and stage-specific expression of proliferating cell nuclear antigen (PCNA) from the malarial parasite, Plasmodium falciparum.

The gene encoding the malarial homologue of proliferating cell nuclear antigen, PCNA, has been identified and characterised. It is located on chromosome 13. The coding sequence of 825 nucleotides predicts a protein of 30,586 Da. There are no introns and northern analysis reveals a transcript of approximately 1.6kb. The conserved residues which characterise the PCNAs of human, Drosophila, Saccharomyces and Xenopus are present in PfPCNA but the overall identity of PfPCNA with human and yeast PCNAs is low; 34% and 31% respectively. PfPCNA is longer than the PCNAs of these other species by about 16 amino acids, most of which are present in a block near the carboxy terminus. Antibodies against a purified PfPCNA-glutathione-S-transferase fusion protein recognise a single band in western blots of parasite extracts at 32kDa. The same antiserum has been used to demonstrate that the expression of PfPCNA is regulated during the intraerythrocytic development of the parasite. Expression increases dramatically in late trophozoites and is maintained during the subsequent nuclear divisions which produce schizonts.

Pyrimethamine resistant mutations in Plasmodium falciparum.

Three mutations in Plasmodium falciparum yielding increased resistance to pyrimethamine were obtained following treatment with chemical mutagens and selection in presence of pyrimethamine. From parasite clone TM4/8.2 a mutant, TM4/8.2/4.1, was produced which raised pyrimethamine resistance about 500 times and was found to involve an amino acid change in the DHFR-TS enzyme molecule from Ser108 to Asn108. A clone of another isolate, T9/94, yielded a mutant, T9/94/300.300, raising pyrimethamine resistance about 10 times and involving an amino acid change from Ile164 to Met164. However, another mutant from T9/94, T9/94/M1-1(b3), although it raised the pyrimethamine resistance 100 times, did not involve any changes in the coding sequence of the DHFR-TS gene, but resulted in the production of about twice as much DHFR-TS enzyme as the original clone T9/94. No amplification of the DHFR-TS gene was detected. It is concluded that changes in pyrimethamine resistance of malaria parasites may arise in at least 2 ways: (1) by structural changes in the DHFR domain of the DHFR-TS gene (as previously found by other workers); (2) by other changes, possibly affecting the expression of the DHFR-TS gene. The relative importance of these 2 mechanisms in causing resistance in wild populations of P. falciparum is discussed.

DNA polymerase delta: gene sequences from Plasmodium falciparum indicate that this enzyme is more highly conserved than DNA polymerase alpha.

Genes encoding proteins homologous to the catalytic subunits of DNA polymerase alpha and delta have been cloned from the human malaria parasite Plasmodium falciparum. These are among the first cellular replicative DNA polymerase genes to be cloned and their sequences allow us to make new statements about the relative degrees of conservation of these two enzymes. The most important finding was that P. falciparum Pol delta showed considerable homology to the only other Pol delta enzyme for which published sequence is available, that of S. cerevisiae, displaying an overall amino acid identity of 45% and identity over a highly conserved central region of 59%. In contrast, the level of identity shown over the equivalent central region of Pol alpha between the P. falciparum and S. cerevisiae sequences is only 32%. The sequence data also allowed us to examine the degree of conservation in putative exonuclease domains of Pol delta. The Pol delta gene of P. falciparum maps to chromosome 10 and evidence is presented for the presence of different sized Pol delta mRNA's in the asexual and sexual erythrocytic stages of parasite development.

An exported protein of Plasmodium falciparum is synthesized as an integral membrane protein.

Exp-1 is an antigen of Plasmodium falciparum which is transported from the parasite cell to the membrane of the parasitophorous vacuole and to membranous compartments in the erythrocyte. To investigate how this protein is transported, we studied the synthesis and membrane translocation of exp-1 in a cell-free system. The protein was translocated into canine pancreatic microsomes. Its N-terminal half was thus protected from proteinase K digestion, suggesting that exp-1 is an integral membrane protein with its N-terminus facing the lumen of the microsomes. This conclusion has been confirmed in vivo. In parasitized erythrocytes, exp-1 is membrane-associated and resistant to extraction with alkali, as would be expected for an integral membrane protein. Moreover, using segment-specific monoclonal antibodies, we have shown that here again the N-terminus of exp-1 faces the inside of vesicles, inaccessible to proteases, whereas the C-terminus is degraded. We conclude that exp-1 is an integral membrane protein and infer that it is transported by vesicles from the parasite to a compartment in the host cell cytoplasm.

The complete sequence of a Plasmodium malariae SSUrRNA gene and its comparison to other plasmodial SSUrRNA genes.

A gene encoding the small subunit rRNA (SSUrRNA) has been isolated from the human parasite, Plasmodium malariae. The gene has been sequenced. It contains conserved and variable regions which conform to patterns established for other eukaryotic SSUrRNA genes. Comparisons with other SSUrRNA genes from Plasmodium species reveal regions unique to P. malariae which could be used in specific diagnostic probes for this organism, and provide evidence that the gene is of the type expressed during asexual growth. In addition the '5.8S' gene has been cloned from P. malariae. The gene has been sequenced. It contains bases universally conserved in '5.8S' genes but there is considerable divergence between the P. malariae sequence and that of the P. falciparum gene.

Expression of alpha and beta tubulin genes during the asexual and sexual blood stages of Plasmodium falciparum.

Malaria parasites switch to sexual development after a period of vegetative growth in the host's erythrocytes. This switch, vital for parasite transmission to mosquitoes, is little understood at the genetic level. Likely candidates for developmental control are the alpha- and beta-tubulin subunits required for microtubule assembly. We report here that the transcription of the alpha- and beta-tubulin genes in Plasmodium falciparum show a radically different pattern of transcription in the sexual and sexual phases of parasite growth. Our studies lead to the conclusion that three transcripts of the beta-tubulin gene differ by sequences in their 5'- or 3'-untranslated regions.

Characterisation and sequence of a protective rhoptry antigen from Plasmodium falciparum.

We have recently demonstrated that a non-polymorphic rhoptry antigen, RAP-1 (rhoptry associated protein-1), which is recognised by human immune serum, can successfully protect Saimiri monkeys from a lethal infection of Plasmodium falciparum malaria. In this report we further characterise the antigen, which consists of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa, and present the antigen's gene sequence. Monoclonal antibody evidence, autocatalytic processing and immunological cross-reactivity suggest that all components of this antigen are derived from the same precursor protein. The antigen is lipophilic, and disulphide bonding plays an important role in its structure. We discuss the structure and function of RAP-1 in the light of its deduced amino acid sequence and consider the relationship of this antigen to other rhoptry antigens of similar subunit size and composition.

Cloning of a beta-tubulin gene from Plasmodium falciparum.

We describe the isolation and characterization of a gene for beta-tubulin from the malaria parasite, Plasmodium falciparum. This organism appears to contain a single gene encoding beta-tubulin. A single transcript from this gene can be detected in the total RNA of the parasite's asexual blood stages. The complete sequence for the gene has been elucidated. It has two introns, one of which has a position identical to that of a related parasite, Toxoplasma gondii. The gene shows the usual preference for codons with A or T in the third position. The predicted amino acid sequence is compared with that of T. gondii and the human host. Further comparisons between these and fungal sequences of beta-tubulins resistant to benomyl, a drug binding this protein, highlight differences that could be exploited in the development of parasite-specific antitubulin drugs.

Identification of Plasmodium falciparum-infected mosquitoes using a probe containing repetitive DNA.

A cloned repetitive DNA sequence (rep20) was evaluated as a diagnostic probe specific for Plasmodium falciparum sporozoites using experimentally infected mosquitoes squashed directly on nylon filters. Head/thorax portions of mosquitoes, killed 14-16 days after ingesting P. falciparum-infected blood, gave positive signals when examined for the presence of P. falciparum sporozoite DNA by hybridisation. This correlated with the number of oocysts found in a sample of the same batch of mosquitoes examined by dissection. No positive signals were obtained with 50 Plasmodium berghei-infected mosquitoes probed with the rep20 sequence. The results indicate that a probe containing rep20 may be useful in the rapid and specific incrimination of vectors carrying P. falciparum sporozoites. The value of repetitive DNA in the diagnosis of malaria is discussed.

Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum.

Merozoites of the malaria parasite Plasmodium falciparum carry surface proteins processed from a precursor termed p190 or p195. Polymorphism has been reported in this protein. Since the protein is a candidate for a malaria vaccine, it is important to understand the nature of this polymorphism. We have determined the complete nucleotide sequence of the p190 gene from the MAD20 strain (a Papua New Guinea isolate). Comparisons of the gene with that from other strains of P. falciparum allowed us to study the genetic basis of the antigen's polymorphism. The gene consists of sequences distributed in variable blocks, which are separated by conserved or semi-conserved sequences. Variable sequences occur both in regions that code for tripeptide repeats and in regions with no apparent repeats. Interestingly, according to the present data, variable sequences are not widely polymorphic but fall into two distinct types. We argue that the p190 protein is encoded by dimorphic alleles capable of limited genetic exchange and present evidence at the nucleotide level documenting intragenic recombination in Plasmodium.

Characterisation of a repetitive DNA sequence from the malaria parasite, Plasmodium falciparum.

A repetitive DNA fragment cloned from the malaria parasite, Plasmodium falciparum, has been analysed. It contains a 21 base pair sequence which occurs in multiple tandem repeats. Two clusters of the same repeat are found in opposite orientations on the same DNA fragment. The repetitive DNA provides an additional way to distinguish between different strains of parasite by hybridisation to genomic blots and may serve as a species-specific probe for diagnosis.

Polymorphism of the precursor for the major surface antigens of Plasmodium falciparum merozoites: studies at the genetic level.

The gene for the precursor of Plasmodium falciparum merozoite surface antigens has been cloned. The entire sequence of the gene from a Thai isolate of the parasite is reported. It provides evidence for a signal peptide, a region containing short repeating peptides and an anchor sequence. In addition, the 5' end of a Papua New Guinea isolate has been sequenced. Comparison of these and other sequences defines, at the genetic level, a polymorphic region in the protein, and suggests that other parts of the protein are less susceptible to variation. Furthermore it appears that several signal peptides of P. falciparum exhibit extensive sequence homologies.

Cloning studies on the gene coding for L-(+)-lactate dehydrogenase of Plasmodium falciparum.

We show that the L-(+)-lactate dehydrogenase (EC 1.1.1.27;L-lactate: NAD+-oxidoreductase) of Plasmodium falciparum (LDH-P) is encoded in the parasite genome. A monoclonal antibody (McAb 7.2) has been shown to bind the LDH-P subunit which has an apparent molecular mass of 35 kDa. A polyclonal antiserum raised against affinity purified LDH-P has been used to isolate cDNA clones containing LDH-P epitopes from a lambda gt11Tn5 expression library. DNA sequence analysis of one clone, lambda LDH-P.1, reveals a single open reading frame which shows a degree of homology to the N-terminal domain of known LDH amino acid sequences.

The gene for an exported antigen of the malaria parasite Plasmodium falciparum cloned and expressed in Escherichia coli.

An exported protein of the erythrocytic stages of the malaria parasite, Plasmodium falciparum, has epitope(s) in common with the surface of the sporozoite stage (1). Two cDNA clones encoding this protein, Ag5.1, have now been isolated and expressed in Escherichia coli. The coding sequence contains a region with strong homology to that of the circumsporozoite protein of P. falciparum. Other features of the sequence can be explained in terms of the observed behaviour of the protein in the parasite life cycle. The Ag5.1 can now be synthesised in bacteria in sufficient amounts to analyse the immune response to this protein.

Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria.

The late blood stages of the human malaria parasite, Plasmodium falciparum, carry a major surface antigen, p190, of molecular weight (Mr) 190,000. This antigenically variable protein is actively processed, first as the parasite matures and again when it is released into the blood stream and invades a new erythrocyte to initiate a cycle of growth. It elicits a strong immune response in man; all tested adult sera from endemic areas have antibodies against this protein. Our evidence indicates that purified p190 can alter the course of parasitaemia in monkeys with falciparum malaria. We have also succeeded in cloning part of the gene for p190 and expressing it in Escherichia coli. To this end we have developed a new technique, antibody select, which greatly simplifies final identification of expressing clones.

Characterisation and translation studies of messenger RNA from the human malaria parasite Plasmodium falciparum and construction of a cDNA library.

RNA was isolated from trophozoites, schizonts and mixed populations of Plasmodium falciparum. 5% of the total was poly(A+) message, of average length 1.2 kb (10-12 kb maximum) and a poly(A) content of 10%. The mRNA fractions could be translated in vitro by reticulocyte lysates supplemented either with exogenous or P. falciparum tRNA. The patterns from two independent isolates, one cloned (T9-94) and one uncloned (K1) were virtually identical. Major translation products from 16-230 kDa have been measured. The most abundant is lactate dehydrogenase (34.8 kDa). Trophozoite mRNA codes principally for proteins of less than or equal to 93 kDa, while schizont mRNA codes for additional proteins of higher molecular mass. There are marked similarities between the in vitro translation products and proteins synthesised in vivo in synchronous cultures. A number of schizont mRNA translation products (principally those of 230, 203, 185, 170, 115, 101 and 71 kDa) are specifically precipitated without post-translational modification by sera from humans exposed to malaria. A cDNA library has been constructed in phage lambda from total poly(A+) RNA and partially characterised. About 10% of the clones derive from abundant mRNA sequences. Putative actin clones have been isolated from this library and the parasite actin mRNA sized at approx. 2.8 kb.