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U Certa

Publications and source records attributed to U Certa.

At least 37 records · Page 2Linked to original sources
PubMed1998

Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays.

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of selected genes. This technology allows simultaneous and quantitative measurement of the transcriptional activity of entire bacterial genomes on a single oligonucleotide probe array.

Biotinylation↗
PubMed1997

Expression and characterisation of plasmepsin I from Plasmodium falciparum.

Two aspartic proteinases, plasmepsins I and II, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of Lys110P to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5-10-fold lower k(cat)/Km values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P. falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin II. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of P. falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.

Animals↗
PubMed1996

Efficient control of gene expression by single step integration of the tetracycline system in transgenic mice.

Tetracycline-regulated gene expression in eukaryotic cell lines, plants, and transgenic mice has become a powerful tool for the analysis of eukaryotic gene expression and function. The system consists of two plasmids, one encoding the transactivator protein under control of a viral cytomegalovirus promoter, and the second being the tet-operator minimal promoter driving the gene of interest. Here we show that these control elements, when integrated in cis on a single plasmid, allow efficient and tight control of reporter gene expression in vitro and in vivo. Dependent on the route of administration of tetracycline, gene expression can be partially or fully repressed in transgenic mice, whereas removal of the antibiotic induces the reporter gene in various tissues to levels up to 800-fold more than the two-plasmid system. In addition, crossing and analysis of animals transgenic for the individual components of the system are unnecessary, and genetic segregation of the control elements during breeding is prevented.

Animals↗
PubMed1994

Cloning and characterization of a novel Plasmodium falciparum sporozoite surface antigen, STARP.

A novel Plasmodium falciparum sporozoite antigen, STARP (Sporozoite Threonine and Asparagine-Rich Protein), detected consistently on the surface of sporozoites obtained from laboratory strains and field isolates, has been identified and cloned, following a systematic approach aimed at isolating novel non-CS sporozoite surface antigens. The 2.0-kb STARP gene has a 5' miniexon/large central exon structure and contains a complex repetitive region encoding multiple dispersed motifs and tandem 45- and 10-amino acid repeats. In sporozoites, transcription of the STARP gene has been conclusively demonstrated by reverse PCR and Northern blot hybridisation and the 78-kDa protein has been localized by immunofluorescence and immunoelectron microscopy to the sporozoite surface. STARP is also expressed in liver stages, as revealed by immunofluorescence assays using antisera raised either to the central repetitive region or the C-terminal non-repetitive region. Expression is also detected in early ring stages, though not in mature erythrocytic or sexual stages. Identification and elucidation of this novel antigen is a step forward in current efforts aimed at developing an effective preerythrocytic-stage malaria vaccine.

Amino Acid Sequence↗
PubMed1994

Regular initiation of translation of Plasmodium berghei aldolase-2 after pre-mRNA splicing.

In Plasmodium falciparum aldolase a UAG or a regular AUG codon has been proposed for the initiation of ribosomal protein synthesis. A UAG codon present at the beginning of the coding sequence of the aldolase 2 gene (aldo-2) of Plasmodium berghei is not recognised in vitro as an initiation codon, which suggests addition of a regular AUG codon by mRNA splicing. Sequence analysis of cDNA amplified by the reversed polymerase chain reaction reveals addition of an ATG codon with a splice donor consensus sequence to the aldo-2 exon. By the same technique and northern blot analysis, substantial amounts of partially spliced P. berghei aldo-2 precursor mRNA are detected which could explain the isolation of immature P. falciparum aldolase cDNA clones starting with a stop codon.

Animals↗
PubMed1993

A conserved region of the MSP-1 surface protein of Plasmodium falciparum contains a recognition sequence for erythrocyte spectrin.

The major surface protein MSP-1 of Plasmodium falciparum blood-stage malaria parasites contains notably conserved sequence blocks with unknown function. The recombinant protein 190L, which represents such a block, exhibits a high affinity for red blood cell membranes. We demonstrate that both 190L and native MSP-1 protein bind to the inner red blood cell membrane skeleton protein spectrin. By using overlapping peptides covering the 190L molecule, we show that the spectrin contact site of 190L is included in a linear sequence of 30 amino acid residues. Association of 190L with naturally occurring spectrin deficient red blood cells is drastically reduced. In the same cells parasite invasion is normal, but the intracellular parasite development arrests late in the trophozoite stage. A similar situation arises when synthetic peptides covering the spectrin recognition sequence of 190L are added to P.falciparum cultures. These data and the cellular localization of MSP-1 suggest the possibility that MSP-1 associates with spectrin under natural conditions.

Amino Acid Sequence↗
PubMed1993

Selective inhibition of Plasmodium falciparum aldolase by a tubulin derived peptide and identification of the binding site.

Aldolase of the human malaria parasite Plasmodium falciparum (PfAldo) may be a potential target for the development of novel antimalarial drugs. Using in vitro mutagenesis we analyzed the function of the carboxy-terminus of the recombinant enzyme. Deletion of the carboxy-terminus of PfAldo confirmed its critical role in catalysis; exchange of conserved residues minimally affected enzyme activity. We exchanged a pair of parasite specific lysine residues with corresponding amino acids of the host. These mutant enzymes exhibited an increased catalytic activity and reduced binding to erythrocyte band 3 protein. Homologous peptides of human band 3 protein and P. falciparum alpha-tubulin were competitive inhibitors of PfAldo. Selective inhibition of PfAldo by the alpha-tubulin peptide depends on the presence of tandem lysine residues and the fine structure of the inhibitor peptide. Our data support the concept of a matrix organisation of glycolytic enzymes in Plasmodium falciparum.

Amino Acid Sequence↗
PubMed1992

Plasmodium falciparum: the repetitive MSA-1 surface protein of the RO-71 isolate is recognized by mouse antibody against the nonrepetitive repeat block of RO-33.

We have expressed in Escherichia coli the nonrepetitive repeat zone of the MSA-1 surface protein of the RO-33 isolate of Plasmodium falciparum. The recombinant protein was used to immunize mice and the resulting RO-33 monospecific serum was used to screen our P. falciparum strain collection in order to recover additional alleles lacking tripeptide repeats in block 2 of MSA-1. Only 1 (RO-71) out of 30 isolates tested reacted strongly with the serum by indirect immunofluorescence assay. Surprisingly, block 2 of the RO-71 MSA-1 allele contains tripeptide repeats resembling those of the K1 isolate of P. falciparum. Additional sequence analysis of the entire DNA coding for the 80-kDa MSA-1-derived surface component did not reveal any amino acid stretches similar to block 2 of RO-33 which could rationalize the immunological cross-reactivity. We eliminated the possibility that the RO-71 culture was contaminated with RO-33 type alleles of MSA-1 by Southern blotting and PCR analysis. The RO-33-specific mouse serum used for the initial selection of RO-71 did not react with the antigen in the denatured state (Western blot). This and the sequence analysis suggest that the cross-reactive epitope in the MSA-1 protein of RO-71 is conformational. The possibility that a truncated frame-shift protein encoded by mutated MSA-1 mRNA is recognized by the serum is discussed.

Amino Acid Sequence↗
PubMed1992

Stage-specific expression of aldolase isoenzymes in the rodent malaria parasite Plasmodium berghei.

We have cloned two gene (aldo-1 and aldo-2) encoding the glycolytic enzyme aldolase of the rodent malaria parasite Plasmodium berghei. The amino acid sequence of one gene product, ALDO-1, is virtually identical to P. falciparum aldolase whereas ALDO-2, the second gene product, is different and has 13% sequence diversity to ALDO-1. We expressed ALDO-2 as an active enzyme in Escherichia coli and compared the biochemical and kinetic properties to that of P. falciparum recombinant aldolase (ALDO-1 type). Based on the Km and Vmax constants for FMP and FBP, neither ALDO-1 nor ALDO-2 can be clearly assigned to any of the known mammalian isoenzyme classes. We demonstrate that expression of the two isoenzymes is developmentally regulated: specific antibody probes detect ALDO-1 in sporozoite stages of P. berghei and ALDO-2 is found in blood stage parasites.

Amino Acid Sequence↗
PubMed1992

Identification and purification of glucose phosphate isomerase of Plasmodium falciparum.

The multiplication of malaria parasites within red blood cells is energy dependent. Since these parasites lack a functional tricarboxylic acid cycle, the energy needs of the parasite are met by anaerobic glycolysis of exogenous glucose. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase, phosphoglycerate kinase and pyruvate kinase have been detected in infected erythrocytes. Here we report a 4-9 times increase in glucose phosphate isomerase (GPI) activity of infected erythrocytes over that of normal erythrocytes. This increase is of parasitic origin, as additional enzyme bands were observed in lysates of infected erythrocytes. The expression of GPI parallels parasite maturation and reaches a maximum at the trophozoite/schizont stage. Two distinct but closely related activity patterns consisting of 3-4 GPI isoenzymes (not shown in normal erythrocytes) with neutral to weakly acidic isoelectric points were observed in 6 P. falciparum isolates tested by isoelectric focusing. The purified P. falciparum GPI has an apparent size of 66 kDa. No size variation was observed in the 6 P. falciparum isolates studied. Furthermore, antiserum raised against this protein in BALB/c mice specifically inhibits parasite encoded GPI activity while no effect was observed on host enzyme activity.

Animals↗
PubMed1992

Molecular analysis of Plasmodium falciparum hexokinase.

Hexokinase, a key glycolytic enzyme, is involved in the initial phosphorylation reaction of imported glucose and specific blocking of this activity may therefore arrest the development of malaria parasites. We describe here the cloning of a single copy hexokinase gene of Plasmodium falciparum (PfHK) from cDNA or genomic DNA libraries. The deduced amino acid sequence of PfHK has 26% identity with human hexokinase I and its predicted molecular mass assigns it as an invertebrate type isoenzyme of hexokinase. A single 1.5-kb exon is translated from a 3-kb mRNA in asexual stages of the parasite. In contrast to aldolase and GPI, the gene for this glycolytic enzyme is located on chromosome 8. Poly- and monoclonal antibodies against recombinant PfHK support our cloning results at the protein level as they detect a protein of the predicted size and isoelectric point by Western blotting in parasite protein samples. Moreover, polyclonal rabbit IgG against recombinant PfHK partially inhibits the hexokinase activity of a P. falciparum lysate which provides direct proof that the gene cloned encodes hexokinase of the parasite.

Amino Acid Sequence↗
PubMed1992

Protection against malaria in Aotus monkeys immunized with a recombinant blood-stage antigen fused to a universal T-cell epitope: correlation of serum gamma interferon levels with protection.

The major surface antigen p190 of the human malaria parasite Plasmodium falciparum contains nonpolymorphic, immunogenic stretches of amino acids which are attractive components for a subunit vaccine against malaria. One such polypeptide, termed 190L, is contained in the 80-kDa processing product of p190, which constitutes the major coat component of mature merozoites. We report here that immunization of Aotus monkeys with 190L gives only poor protection against P. falciparum challenge. However, addition by genetic engineering of a universal T-cell epitope (CS.T3) to 190L improved immunity, and as a result three of four monkeys were protected following challenge infection with blood-stage parasites. Neither antibody against the immunizing antigens or against blood-stage parasites nor the capacity of the monkeys' sera to inhibit in vitro parasite invasion correlated with protection. However, in contrast to sera from nonprotected monkeys, sera from protected animals contained elevated levels of gamma interferon. These results suggest that gamma interferon is directly or indirectly involved in the process of asexual parasite control in vivo.

Animals↗
PubMed1992

Efficiency of human Plasmodium falciparum malaria vaccine candidates in Aotus lemurinus monkeys.

The protective efficacy of several recombinant and a synthetic Plasmodium falciparum protein was assessed in Aotus monkeys. The rp41 aldolase, the 190L fragment of the MSA-1 protein and fusion 190L-CS. T3 protein containing the CS.T3 helper "universal" epitope were emulsified in Freund's adjuvants and injected 3 times in groups of 4-5 monkeys each one. The synthetic polymer Spf (66)30 also emulsified in Freund's adjuvants was injected 6 times. Control groups for both experiments were immunized with saline solution in the same adjuvant following the same schedules. Serology for malaria specific antibodies showed seroconversion in monkeys immunized with the recombinant proteins but not in those immunized with the polymer nor in the controls. Challenge was performed with the 10(5) parasites from the P. falciparum FVO isolate. Neither rp41 nor Spf(66)30 induced protection, whereas 190L induced significant delay of parasitemia. The fusion of the CS.T3 epitope to 190L significantly increased its protective capacity.

Animals↗
PubMed1991

Malaria vaccine.

Among infectious diseases caused by protozoa, malaria is still the greatest killer of children. Mortality in adults living in endemic areas is significantly lower because they frequently acquire partial or complete immunity to the major pathogen, Plasmodium falciparum. This natural protection indicates that vaccination may be possible, and the first candidate antigens were cloned with the use of human immune sera as probes. Genetic and biochemical analysis of the parasite proteins revealed that they are polymorphic, and frequently gene sequences were discovered which were specific for a particular parasite isolate, which eliminated most antigens for purposes of vaccine development. The most promising candidate antigens today are the major surface proteins of sporozoites and blood stage parasites. However, the immune response against those is not sufficient for complete protection, and additional, intensive research is necessary to identify new molecules to be included in a vaccine cocktail against malaria. The current spread of the disease due to increasing drug resistance of parasites and mosquito vectors emphasizes the urgent need for a vaccine.

Animals↗
PubMed1991

Is Plasmodium falciparum aldolase useful for rational drug design?

P. falciparum lacks a functional citric acid cycle. Unlike most tissues of the mammalian host, it is totally dependent on glycolysis for energy generation. A compound which selectively inhibits the parasite's ATP-generating machinery is therefore a potential antimalarial agent. Such a drug may interact in two ways: a) by inhibiting the activity of an enzyme or b) by disturbing the micro-organization of consecutive enzymes in a metabolic pathway. In mammalian tissues the glycolytic pathway involves the cytoskeleton as a matrix to keep phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase in an optimal sterical position for rapid substrate conversion. For instance, these three enzymes bind to the band 3 protein in erythrocytes or to actin in muscle cells. P. falciparum aldolase binds with very high affinity to the band 3 protein of human erythrocyte ghosts. However, the true in vivo site of association is believed to be actin II of P. falciparum. This actin has a sequence element which is almost identical to that of the band 3 aldolase binding site. We therefore suppose that plasmodia exploit a similar matrix organization. If true, the association of these enzymes with the cytoskeleton is a target for novel antimalarials. In contrast to all vertebrate aldolases, P. falciparum and P. berghei aldolases have two neighbouring lysine residues near the carboxy-terminus. We show here that mutagenesis of these basic residues has an effect on the catalytic constants Vmax and KM and moreover, the ability to bind to band 3 is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗