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T Triglia

Publications and source records attributed to T Triglia.

At least 19 recordsLinked to original sources
PubMed1998

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria.

We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.

Alleles↗
PubMed1997

Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria in humans. An important class of drugs in malaria treatment is the sulfone/sulfonamide group, of which sulfadoxine is the most commonly used. The target of sulfadoxine is the enzyme dihydropteroate synthase (DHPS), and sequencing of the DHPS gene has identified amino acid differences that may be involved in the mechanism of resistance to this drug. In this study we have sequenced the DHPS gene in 10 isolates from Thailand and identified a new allele of DHPS that has a previously unidentified amino acid difference. We have expressed eight alleles of P. falciparum PPPK-DHPS in Escherichia coli and purified the functional enzymes to homogeneity. Strikingly, the Ki for sulfadoxine varies by almost three orders of magnitude from 0.14 microM for the DHPS allele from sensitive isolates to 112 microM for an enzyme expressed in a highly resistant isolate. Comparison of the Ki of different sulfonamides and the sulfone dapsone has suggested that the amino acid differences in DHPS would confer cross-resistance to these compounds. These results show that the amino acid differences in the DHPS enzyme of sulfadoxine-resistant isolates of P. falciparum are central to the mechanism of resistance to sulfones and sulfonamides.

Animals↗
PubMed1997

Stable transgene expression in Plasmodium falciparum.

Plasmid vectors designed to express transgenes and a selectable marker in Plasmodiumfalciparum were constructed. These consist of a selectable gene cassette comprising the Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene mutated to confer pyrimethamine resistance flanked by either Plasmodium chabaudi DHFR-TS or P. falciparum calmodulin promoter sequences and the P. falciparum histidine rich protein 2 3' region. Also, each vector includes a different expression cassette driven by various Plasmodium transcriptional control sequences. Initially, the chloramphenicol acetyl transferase (CAT) reporter gene was cloned into the expression site of two vectors, pCC6-CAT and pCC13-CAT, which were identical except for the orientation of the expression cassette with respect to the selectable gene cassette. Approximately 8-fold more CAT activity was detected when the direction of transcription of the expression cassettes was in a head to head, rather than a tail to head, orientation. Importantly, it was found that stable transfection could only be achieved when the gene cassettes were in the head to head direction suggesting that this orientation also has an effect on the level of expression of the selectable marker. All other plasmids were designed with the cassettes in a head to head orientation. With the exception of pCC6-CAT and a second vector pHC4-CAT, stable transfectants were obtained with each vector in which the CAT gene had been inserted into the expression cassette. This is the first time vectors for the stable expression in Plasmodium parasites of transgenes other than a selectable marker have been described.

Animals↗
PubMed1995

A YAC contig map of Plasmodium falciparum chromosome 4: characterization of a DNA amplification between two recently separated isolates.

We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen.

Animals↗
PubMed1995

Molecular cloning of a gene from Plasmodium falciparum that codes for a protein sharing motifs found in adhesive molecules from mammals and plasmodia.

Adhesion of Plasmodium to host cells is an important phenomenon in parasite invasion and in malaria-associated pathology. We report here the molecular cloning of a putative adhesive molecule from P. falciparum that shares both sequence and structural similarities with a sporozoite surface molecule from Plasmodium termed the thrombospondin-related anonymous protein (TRAP) and, to a lesser extent, with the circumsporozoite (CS) protein. The gene, which is present on chromosome 3 as a single copy, was termed CTRP for CS protein-TRAP-related protein. The full-length CTRP encodes a protein containing a putative signal sequence followed by a long extracellular region of 1990 amino acids, a transmembrane domain, and a short cytoplasmic segment. The putative extracellular region of CTRP is defined by two separated adhesive domains. The first domain contains six 210-amino acid-long homologous repeats, the sequence of which is related to the A-type domain found in adhesive molecules including the alpha subunits of several integrins and a number of extracellular matrix glycoproteins. The second domain contains seven repeats of 87-60 amino acids in length, which share similarities with the thrombospondin type 1 domain found in a variety of adhesive molecules. Finally, CTRP also contains consensus motifs found in the superfamily of haematopoietin receptors. Interstrain analysis of eight different parasite isolates revealed that CTRP does not show size polymorphism except in repetitive regions flanking potential adhesive domains.

Amino Acid Sequence↗
PubMed1994

Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum.

The enzyme dihydropteroate synthetase (DHPS) from Plasmodium falciparum is involved in the mechanism of action of the sulfone/sulfonamide group of drugs. We describe the cloning and sequencing of the gene encoding the P. falciparum DHPS enzyme and show that it is a bifunctional enzyme that includes dihydro-6-hydroxymethylpterin pyrophosphokinase (PPPK) at the N terminus of DHPS. The gene encodes a putative protein of 83 kDa that contains two domains that are homologous with the DHPS and PPPK enzymes of other organisms. The PPPK-DHPS gene is encoded on chromosome 8 and has two introns. An antibody raised to the PPPK region of the protein was found to recognize a 68-kDa protein that is expressed throughout the asexual life cycle of the parasite. We have determined the sequence of the DHPS portion of the gene from sulfadoxine-sensitive and -resistant P. falciparum clones and identified sequence differences that may have a role in sulfone/sulfonamide resistance.

Amino Acid Sequence↗
PubMed1994

Mapping the genetic locus implicated in cytoadherence of Plasmodium falciparum to melanoma cells.

Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro cultivation accompanied by loss of cytoadherence to melanoma cells. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling PfEMP1 expression is encoded in this region. Initially a library of short fragments highly enriched for the right arm of chromosome 9 was constructed in bacteriophage lambda. Clones from this library were obtained randomly by the polymerase chain reaction (PCR) technique, sequenced and used to screen a yeast artificial chromosome (YAC)-P. falciparum library by PCR so that the region could be cloned and physically mapped in detail. We have used probes from this region to demonstrate that clones derived from ITG2 have undergone a deletion of intermediate length on chromosome 9. This could explain the unusual stability of cytoadherence in these clones.

Animals↗
PubMed1994

Relationship between humoral response to Plasmodium falciparum merozoite surface antigen-2 and malaria morbidity in a highly endemic area of Papua New Guinea.

The prevalence and concentration of antibodies to merozoite surface antigen-2 (MSA-2) were measured in blood samples collected during a cross-sectional survey. Antibodies were measured by enzyme-linked immunosorbent assay using two recombinant proteins that closely approximated the full-length mature MSA-2 polypeptides expressed by the Plasmodium falciparum isolate FC27 and the cloned line 3D7 and that were representative of the dimorphic forms of MSA-2. Antibodies were also measured to a form of the 3D7 MSA-2 lacking the central repetitive sequences (d3D7). High antibody prevalence was observed to all three antigens: the overall prevalence of IgG to FC27, 3D7, and d3D7 was 91%, 90%, and 90%, respectively. The majority of individuals > or = 5 years of age had antibodies to both forms of MSA-2. The geometric mean antibody units increased with age with a plateau being reached by 15-20 years of age. There was a significant positive association of antibody prevalence with both the presence of the parasite and an enlarged spleen in children. This study provides the first evidence that antibodies against nonrepeat regions of MSA-2 are associated with fewer fever episodes and less anemia, both known to be indicators of malaria morbidity.

Adolescent↗
PubMed1992

A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence.

Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis. Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and the selected cells produce gametocytes. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region. We have isolated several markers for the deleted region and are currently using a YAC-P. falciparum library to investigate this region of the genome in detail.

Animals↗
PubMed1991

Large fragments of Plasmodium falciparum DNA can be stable when cloned in yeast artificial chromosomes.

A major problem in cloning large segments of Plasmodium falciparum DNA is the instability of this very A + T-rich DNA in Escherichia coli. We have therefore investigated whether P. falciparum DNA segments over 100 kb in size cloned in the yeast Saccharomyces cerevisiae as yeast artificial chromosomes (YACs) are stable. An analysis of EcoRI fragments adding up to 230 kb, or nearly 1% of the P. falciparum genome, showed that sequences cloned as YACs were indistinguishable from the DNA in the P. falciparum genome, showing neither deletions nor rearrangements. Over 400 YACs have been obtained so far with an average insert size between 110 and 130 kb. Screening the YACs with single-copy probes suggested that most sequences should be represented in a library of less than 10(3) YACs. Hence YACs provide an approach to cloning large segments of P. falciparum DNA.

Animals↗
PubMed1991

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.

Animals↗
PubMed1987

Fluctuations in the expression of a glycolipid antigen associated with differentiation of melanoma cells monitored by a monoclonal antibody, Leo Mel 3.

A monoclonal antibody, Leo Mel 3, raised against a melanoma cell line (LiBr), binds to a carbohydrate determinant of cell surface gangliosides, the simplest of which is GD3. This monoclonal antibody was screened for by its capacity to block the recognition and lysis of the melanoma cells by cytotoxic T-lymphocytes with anomalous killer cell function, illustrating a novel approach for identifying monoclonal antibody to biologically relevant tumor-associated antigens. Leo Mel 3 reacted selectively with melanoma cells by indirect immunofluorescent and immunoperoxidase staining; it reacted with tissue from all primary and metastatic melanoma tested, and it bound to cells from all but one of six cultured melanoma cell lines. Leo Mel 3 did not react with a variety of carcinomas, lymphomas, leukemias, and other neuroectodermal tumors, nor with adult or fetal tissues, except fetal liver. Very weak staining of cutaneous basal melanocytes was noted in a minority of skin sections, and 50 to 80% of melanocytes in four of seven benign nevi showed weak to moderate reactivity. The antibody was relatively specific for human adherent melanoma cells, since it did not bind to the adherent murine B16 melanoma line nor to a nonadherent human melanoma cell line (PMC-22). Expression of the Leo Mel 3-defined antigen was unrelated to changes in cell cycle. When cells from an adherent melanoma cell line were detached and maintained briefly in suspension culture, the cells became markedly less reactive with Leo Mel 3 and, after readherence to plastic, they rapidly reexpressed higher levels of the ganglioside antigen; since Leo Mel 3 prevented attachment and growth of melanoma cells in vitro, a functional role for the ganglioside is suggested in cell adhesion and metastasis. Differentiation of melanoma cells with dimethyl sulfoxide, retinoic acid, and theophylline resulted in a marked and selective increase in the amount of Leo Mel 3-defined antigen, together with an increase in the target cell binding ability of these cells, assessed by cold target competition assays using anomalous killer cells.

Antibodies, Monoclonal↗
PubMed1987

The complete sequence of the gene for the knob-associated histidine-rich protein from Plasmodium falciparum.

'Knobs' at the surface of erythrocytes infected with mature stages of Plasmodium falciparum are believed to be important in adherence of these cells to capillary walls. They contain at least one parasite protein, designated the knob-associated histidine-rich protein (KAHRP). We present here the sequences of a cDNA and chromosomal clone that predict the complete sequence of KAHRP. The gene contains a single intervening sequence, located at the 3' boundary of the hydrophobic core of a putative signal sequence. Exon two encodes a short region that is rich in histidine as well as two separate regions of repetitive sequence, the 5' repeats (five copies related to SKKHKDNEDAESVK) and the 3' repeats (seven copies related to SKGATKEAST). These repeat blocks were both shown to bear epitopes recognized by the human immune system during natural infection by expressing them separately in Escherichia coli, and reacting human antibodies affinity-purified on lysates of the resulting clones with the corresponding synthetic oligopeptides. The 3' end of the molecule, presumably the repetitive region, is a site of size variation in KAHRP from different isolates.

Amino Acid Sequence↗
PubMed1986

The IIb-IIIa glycoprotein complex that mediates platelet aggregation is directly implicated in leukocyte adhesion.

Evidence is presented that the IIb-IIIa glycoprotein complex, which functions as the receptor for fibrinogen on platelets and is central to platelet aggregation, is expressed on the surface of leukocytes where it may function as a receptor for fibronectin. F(ab')2 fragments of a monoclonal antibody, 25E11, raised against activated large granular lymphocytes, inhibited killing by natural killer cells, blocked the binding of fibronectin-coated particles by monocytes, and stimulated neutrophils to exhibit increased antibody-dependent killing. Immunoprecipitation studies of leukocytes and platelets, and the ability of 25E11 to inhibit platelet aggregation, identified the antigen as an epitope on the IIb-IIIa complex. This glycoprotein thus constitutes the first example of a receptor mediating both platelet aggregation and leukocyte adhesion.

Adult↗
PubMed1986

Cloned human cytotoxic T lymphocytes develop anomalous killer cell function.

Mixed lymphocyte cultures were set up between blood mononuclear cells and irradiated autologous or allogeneic B lymphoblasts infected with Epstein-Barr virus. The resulting cytotoxic effector cells were cloned and tested for activity against the stimulating B lymphoblast, K562 and melanoma targets. Specific clones which killed only the stimulating B lymphoblasts (cytotoxic T lymphocytes; CTL) were re-cloned and the subclones tested for cytolysis of B lymphoblasts and melanoma cells. Of seven primary CTL clones generated in allogeneic culture, 308 subclones developed the ability to kill melanoma cells and none retained specific CTL function. In the autologous system, 180 subclones were derived from three specific primary clones: of these, 13 (7%) retained specific function, 29 (16%) were able to kill both B lymphoblasts and melanoma cells, and 93 (52%) killed only the melanoma target. Testing of random clones demonstrated that whereas both B lymphoblast killing (CTL function) and melanoma cell killing (anomalous killer; AK function) were blocked by a monoclonal antibody to LFA-1, only CTL function was blocked by anti-T3 or anti-T8 antibodies. The factor(s) causing the progression of CTL to AK cells are discussed. These data thus demonstrate that the majority of CTL are capable of mediating AK cell function and are thus potentially suitable for passive immunotherapy.

Antigens↗