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U Certa

Publications and source records attributed to U Certa.

52 records · Page 3Linked to original sources
PubMed1990

Specificity and inhibitory activity of antibodies to Plasmodium falciparum aldolase.

The multiplication of Plasmodium falciparum within RBC is energy-dependent and the glucose consumption of infected RBC is increased more than 50 times over the consumption of normal RBC. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase (p41) have been detected in infected RBC. Expression of the cloned aldolase gene of P. falciparum in Escherichia coli resulted in an enzymatically active polypeptide with a high sp. act. and the recombinant p41 aldolase was used for enzymatic and immunologic studies reported here. The presence of antibodies against p41 in the sera of human adults partially immune to malaria and immunization experiments in monkeys suggest that p41 is implicated in protective immune response against the parasite. Therefore, we analyzed the capacity of various antisera to inhibit P. falciparum aldolase activity. It was found that anti-p41 antibodies raised in mice, rabbits, and monkeys inhibited very efficiently aldolase activity in vitro up to dilutions higher than 10(-3). In contrast none of the human sera with high levels of anti-p41 antibodies were able to inhibit parasite aldolase activity even at a dilution of 1/2. The inability of human antisera to neutralize parasite aldolase is not related to antibody titers but is probably related to the specificity of the human antibodies. This finding is discussed in relation to homology of structure of P. falciparum and mammalian aldolase and to a possible mechanism of parasite adaptation and survival in its natural host.

Animals↗
PubMed1990

Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

The gene coding for the key glycolytic enzyme fructose-1,6-diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.

Amino Acid Sequence↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase.

The energy metabolism of the blood stage form of the human malaria parasite Plasmodium falciparum is adapted to the host cell. Like erythrocytes, P. falciparum merozoites lack a functional citric acid cycle. Generation of ATP depends therefore fully on the glycolytic pathway. Aldolase is a key enzyme of this pathway and a high degree of sequence diversity between parasite and host makes it a potential drug target. We have expressed the enzyme in its tetrameric form in Escherichia coli and the catalytic constants Vmax and Km of the recombinant enzyme correspond to the constants of parasite-derived aldolase. Rabbit antibodies against the recombinant P. falciparum aldolase inhibit the natural enzyme and no cross-reaction with human aldolase is detectable. Both the recombinant and the natural protein bind to the cytosolic domain of the band 3 membrane protein in vitro. A 19-residue synthetic peptide corresponding to the sequence of the binding domain of band 3 is an inhibitor when included in the binding assay. In addition, this peptide inhibits the catalytic activity of recombinant P. falciparum aldolase when assayed in a buffer system devoid of anions such as chloride or phosphate. The band 3-derived peptides compete with the aldolase substrate fructose-1,6-diphosphate for binding, suggesting that both reagents have a high affinity for the substrate pocket. A similar sequence motif exists in P. falciparum actin II. A 19-residue peptide corresponding to this sequence is also an inhibitor which could suggest that the P. falciparum aldolase can associate with the cytoskeleton of the parasite or of the host.

Amino Acid Sequence↗
PubMed1990

Immunization of Aotus monkeys with Plasmodium falciparum blood-stage recombinant proteins.

The current spread of multidrug-resistant malaria demands rapid vaccine development against the major pathogen Plasmodium falciparum. The high quantities of protein required for a worldwide vaccination campaign select recombinant DNA technology as a practical approach for large-scale antigen production. We describe the vaccination of Aotus monkeys with two recombinant blood-stage antigens (recombinant p41 and 190N) that were considered as vaccine candidates because parasite-derived antigen preparations could protect susceptible monkeys from an otherwise lethal malaria infection. In contrast to the natural antigen, recombinant p41 protein (P. falciparum aldolase) could not protect monkeys, although all animals seroconverted. 190N antigen, a recombinant protein containing conserved sequences of the major merozoite surface antigen p190, protected two of five monkeys from critical levels of infection with the highly virulent FVO isolate of P. falciparum. However, the B- and T-cell responses to 190N antigen were similar in protected and unprotected animals so that other unknown factors may contribute to protection. Higher purity or lack of protective epitopes or different structure of protective epitopes in the recombinant proteins might explain the better performance of parasite-derived antigens in vaccination trials. The partial protection obtained with 190N antigen suggests that this molecule could contribute to a vaccine mixture against P. falciparum.

Animals↗
PubMed1989

Requirements for screening recombinant DNA libraries for T cell epitope expression.

The possibility of screening cDNA expression libraries with T cell clones was investigated. The model system was based on human T cell clones specific for the recombinant malaria protein 190L, which was expressed fused to beta-galactosidase in lambda gt11. Several membranes were tested for their capacity to bind antigen and stimulate T cell proliferation. Pretreatment of membranes with DMSO and/or sonication to release the antigen improved the sensitivity of the assay. Under optimal conditions, T cell proliferation in response to antigen bound to a low protein binder membrane was comparable to that observed with the antigen in solution. A dot-blot type apparatus was designed for screening large numbers of plaques with T cells. The technical problems of this approach, its requirements and possible applications are discussed.

Clone Cells↗
PubMed1988

Aldolase activity of a Plasmodium falciparum protein with protective properties.

Immunization with a 41-kilodalton blood stage antigen (p41) of Plasmodium falciparum induces immunity to malaria in monkeys. However, antigenic polymorphism and repetitive amino acids commonly found in protective antigens complicate vaccine development. The gene encoding p41 has now been cloned and analyzed. Sequencing and hybridization studies revealed that the gene structure is highly conserved in 14 parasite isolates from three continents. This finding and the lack of repetitive amino acids in the translated DNA sequence may indicate that p41 has an essential function. In this study the protein was found to be 60 percent homologous to the key glycolytic enzyme aldolase from vertebrates, and the affinity-purified p41 protein from parasites showed aldolase activity.

Animals↗
PubMed1988

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene.

Amino Acid Sequence↗
PubMed1987

A naturally occurring gene encoding the major surface antigen precursor p190 of Plasmodium falciparum lacks tripeptide repeats.

Plasmodium falciparum merozoites have variable surface proteins that are processed from a 190-kd precursor protein (p190). The gene encoding p190 exists in two allelic forms and cross-over events occurring mainly near the 5' end, combined with isolate-specific tripeptide repeats, contribute to its antigen diversity. We have sequenced a large portion of the p190 gene from the parasite isolate RO-33 (Ghana). Remarkably, the typical N-terminal tripeptide repeat structure is lacking. Apart from mutations in the variable parts, the gene appears identical to the MAD-20 allele (Papua, New Guinea). Southern blot analysis detects p190 genes similar to RO-33 in other parasite isolates independent of their geographical origin. The lack of p190 repeats in RO-33 eliminates the possibility that they are involved in host cell recognition or integration and restricts their function to immune escape.

Alleles↗
PubMed1987

Dideoxy sequencing of double-stranded DNA from poly(A)-extended 3'-ends.

A simple method has been developed for sequencing double-stranded DNA by the chain termination method. The DNA to be sequenced is cut with a restriction enzyme that leaves a 3'-overhang which is extended by terminal deoxynucleotidyltransferase with limiting amounts of dATP. The sequencing reaction is then primed with an oligo(dT) primer which has a base pair "anchor" complementary to the overhang generated by the restriction enzyme. The method presented here eliminates the need for subcloning of the DNA or sequencing by chemical modification. Furthermore, sequences of more than 300 nucleotides are obtained from any 3'-overhanging restriction end.

Autoradiography↗
PubMed1986

Subregions of a conserved part of the HIV gp41 transmembrane protein are differentially recognized by antibodies of infected individuals.

A 240-bp DNA fragment encoding a peptide, designated ENV(80), homologous to a conserved part of the gp41 transmembrane glycoprotein of human immunodeficiency virus (HIV) was chemically synthesized and inserted into different plasmid expression vectors. Escherichia coli transformants containing these plasmid constructs produced upon induction high amounts of either an ENV(80) peptide of relative molecular mass (Mr) of 10,000 or the same ENV(80) peptide N-terminally fused to E. coli chloramphenicol acetyltransferase (CAT) or to mouse dihydrofolate reductase (DHFR) having Mr of 36,000 and 31,000 respectively. All polypeptides containing the ENV(80) sequences were strongly reactive with antibodies present in sera from AIDS virus-infected individuals, but not with control sera. The strategy of gene assembly allowed the expression of ENV(80) subfragments fused to DHFR. The serodiagnosis of 15 positive sera by Western blot analysis using these bacterially synthesized ENV(80) subfragments revealed the presence of several immunoreactive epitopes on the 80-amino acid polypeptide which were recognized differently by the various patients.

Acquired Immunodeficiency Syndrome↗
PubMed1985

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.

Amino Acid Sequence↗
PubMed1984

Yeast may not contain histone H1: the only known 'histone H1-like' protein in Saccharomyces cerevisiae is a mitochondrial protein.

It is likely that histone H1 is involved in the condensation of chromatin in eukaryotes. However, both the presence of histone H1 in yeast and the extent of yeast chromatin condensation are controversial. A 20 kD protein copurifies with yeast chromatin and was shown by other investigators to have characteristics of histone H1 protein. In an attempt to obtain a positive identification of the 20 kD protein, we purified the protein to homogeneity and raised antibodies against it. We show here by immunofluorescence that the 20 kD protein does not localize to the nucleus but to cytoplasmic particles resembling mitochondria. Furthermore, we show by Western-blot analysis that anti-20 kD protein antibodies react to protein isolated from purified mitochondria. Finally, we present evidence based on size, charge, amino acid composition and immunological cross reactivity to suggest that the yeast 20 kD protein is likely to be the mitochondrial DNA-binding HM protein. This leaves no candidate for histone H1 in yeast.

Amino Acids↗
PubMedDate not supplied

Recombinant polypeptides for serology of malaria.

We have evaluated 3 molecularly defined polypeptides encoded by encloned Plasmodium falciparum genes for their ability to serve as antigens for detecting antimalaria antibodies. The recombinant proteins correspond to (i) a conserved part of 190-200 kDa schizont merozoite surface component, (ii) the carboxy terminal part of the P. falciparum aldolase, and (iii) the 5.1 antigen. Antibodies were detected using enzyme-linked immunosorbent assays (ELISA) in a high percentage of sera from individuals from a malaria endemic area in The Gambia (up to 99% for some adult groups). These results were further improved, especially for detection of antimalaria antibodies in children, when a pool of all 3 polypeptides (ELISA MIXT) was used as antigen. This ELISA MIXT improves presently available assays for the detection of antimalaria antibodies directed against asexual blood stages in respect of standardization, sensitivity and specificity.

Adult↗