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D F Wirth

Publications and source records attributed to D F Wirth.

At least 37 records · Page 2Linked to original sources
PubMed1999

Host cell proteins bind specifically to the capsid-cleaved 5' end of Leishmaniavirus RNA.

Leishmaniavirus (LRV) is a double-stranded RNA (dsRNA) virus that persistently infects some strains of the protozoan parasite, Leishmania. LRV generates a short transcript, corresponding to the 5' end of the positive-sense RNA (320 nt), via a cleavage event mediated by the viral capsid protein on the full-length positive sense RNA transcript. To address the possibility that the RNA cleavage represents a regulatory mechanism for maintaining persistent infection, the interactions between Leishmania cytoplasmic proteins and in vitro synthesized viral transcripts were studied. In gel mobility shift experiments, three specific RNA/protein complexes were formed between cellular proteins and the cleaved viral transcript, and three major proteins were labeled by UV cross-linking. No protein binding activity was observed for either the short (320 nt) or full-length RNA transcripts. However, the two cleavage reaction products were able to form stable RNA/RNA complexes. We present a model in which the virus is targeting its own transcript for cleavage to promote binding of host factors to cryptic domains inaccessible in the full-length transcript.

5' Untranslated Regions↗
PubMed1999

Isolation and functional characterization of two distinct sexual-stage-specific promoters of the human malaria parasite Plasmodium falciparum.

Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of the pfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activates pfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum.

Animals↗
PubMed1998

Short report: detection of Leishmaniavirus in human biopsy samples of leishmaniasis from Peru.

Leishmaniavirus is a double-stranded RNA virus that persistently infects some strains of the protozoan parasite Leishmania. There is considerable interest in the possibility that the presence of this virus alters parasite phenotype and may affect disease pathogenesis. If so, the virus marker could provide a valuable prognostic indicator for human leishmaniasis, particularly in those cases caused by New World parasite strains. The virus has been detected in cultured L. braziliensis, L. b. guyanensis, and L. major. To date there has been no information as to the extent of infection in samples prior to culturing in the laboratory. This study demonstrates, through the reverse transcription-polymerase chain reaction, that Leishmaniavirus exists in human biopsy samples of leishmaniasis prior to manipulation in culture.

Animals↗
PubMed1998

Characterization of Plasmodium falciparum isolated from the Amazon region of Brazil: evidence for quinine resistance.

The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil. In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P. falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State. All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration [IC50] = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM). The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM). Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively. These point mutations were similar to that previously described in other Brazilian isolates. Southern blot analysis revealed no amplification of the pfmdr1 gene. These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine.

ATP-Binding Cassette Transporters↗
PubMed1995

Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum.

The pfmdr1 gene has been associated with a drug-resistant phenotype in Plasmodium falciparum, and overexpression of pfmdr1 has been associated with mefloquine- and halofantrine-resistant parasites, but little is known about the functional role of pfmdr1 in this process. Here, we demonstrate that the pfmdr1 gene expressed in a heterologous yeast system functions as a transport molecule and complements a mutation in ste6, a gene which encodes a mating pheromone a-factor export molecule. In addition, the pfmdr1 gene containing two mutations which are associated with naturally occurring chloroquine resistance abolishes this mating phenotype, suggesting that these genetic polymorphisms alter this transport function. Our results support the functional role of pfmdr1 as a transport molecule in the mediation of drug resistance and provide an assay system to address the nature of this transport function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Leishmania amazonensis: multidrug resistance in vinblastine-resistant promastigotes is associated with rhodamine 123 efflux, DNA amplification, and RNA overexpression of a Leishmania mdr1 gene.

A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Identification of a cis-acting gene regulatory element from the lemdr1 locus of Leishmania enriettii.

The goal of this work is to identify those elements which control gene expression in the parasitic protozoan, Leishmania enriettii. To date there has been no report of a transcriptional controlling element identified in this parasite. In the present study, we have found that a region of the lemdr1 locus of L. enriettii can down-regulate the steady state mRNA level of the reporter neomycin phosphotransferase gene when it is placed in an extrachromosomal expression vector, pALTneo. Through deletion analysis, we have mapped a putative regulatory sequence to the flanking region upstream of the lemdr1 gene. This cis regulatory element is shown to control gene expression in an orientation-dependent and position independent manner. By performing nuclear run-on analysis, we have demonstrated that this element mainly exerts its effect at the transcriptional level by determining the strandedness of transcription on the pALTneo vector. The identification of this novel cis regulatory element should further our understanding of the transcriptional process in Leishmania and other related trypanosomatids.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Plasmodium falciparum and P. vivax: factors affecting sensitivity and specificity of PCR-based diagnosis of malaria.

We have previously reported on development of a simple PCR-based method to detect Plasmodium falciparum in which patient blood samples are lysed and then filtered onto paper. The present studies, conducted in Thailand, were designed to identify factors contributing to differences between results from microscopy and PCR. To analyze microscope-positive, PCR-negative results, we demonstrated that using the lysis/filtration sample preparation method, target DNA degradation is not a significant factor. Similarly, we showed that sensitivity of PCR among patient samples did not differ using the lysis centrifugation method or organically extracted DNA. We further demonstrated that 7/13 samples which were negative by PCR for P. falciparum were positive by PCR when P. vivax-specific primers were used. Microscope-negative, P. falciparum PCR-positive samples were analyzed in two ways: the true rate of false-positivity for PCR (2%) was established by analyzing 498 samples from patients living in areas without transmission. We further showed that when microscope-negative, PCR-positive samples were amplified using an independent P. falciparum-specific PCR target sequence, 42/47 were PCR-positive. We conclude that the accuracy and reduced limit of detection of microscopy are major confounders when comparing this method to PCR.

Animals↗
PubMed1994

A homologous recombination strategy to analyze the vinblastine resistance property of the V-circle in Leishmania.

The generation of extrachromosomal DNA elements in Leishmania sp. can occur naturally or during in vitro selection with drugs. Previously, we had established a strong association between V-circle amplification and drug resistance in L. enriettii stepwise selected with increasing concentrations of vinblastine. Further, we demonstrated the presence of the lemdr1 gene in the amplified V-circle and subsequent functional analysis by transfection of this gene alone confirmed its role in conferring a drug-resistant phenotype, but the level of resistance was significantly lower than in cell lines obtained from stepwise drug selection. The aim of this work was to determine if other genes either on the V-circle or elsewhere in the genome were necessary for expression of vinblastine resistance. We report here the development of a homologous recombination method to convert the entire V-circle from the LeV160 cell line into a shuttle vector and further the targeted disruption of specific sites within the V-circle. Our results clearly demonstrate that the V-circle alone is sufficient to confer full vinblastine resistance and the disruption of the lemdr1 locus destroys the ability of the V-circle to confer vinblastine resistance.

Animals↗
PubMed1994

DNA probes as epidemiological tools for surveillance of Plasmodium falciparum malaria in Thailand.

We have previously reported on development of a DNA probe-based method for diagnosing Plasmodium falciparum infection directly from patient blood samples. In the present studies, we sought to examine applicability of the method to large epidemiological surveys, comparing sensitivity, specificity, time required to obtain results, and costs with those of conventional microscopic examination. Results of DNA probe hybridization were also compared between laboratories in the US and Thailand, to assess transferability of the DNA probe technology. Five separate surveys of approximately 5000 villagers each were performed between December 1987 and June 1989 (26,176 samples total). Sensitivity ranged from 61% to 92% for both US and Thai laboratories, while specificity ranged from 98.2% to 99.9%. Agreement between the US and Thai laboratories was good, with kappa coefficients between 0.62 and 0.78 for different surveys. Between 4 and 8 person-days were required to obtain results from each set of 5000 samples by DNA hybridization, whereas microscopic examination required 150 person-days. Approximate costs were US 0.17 per sample for DNA probe analysis, and US$0.36 for microscopic examination. We conclude that the DNA probe method offers significant advantages when large numbers of samples must be surveyed for P. falciparum.

Adolescent↗
PubMed1994

Neuronal monoamine reuptake inhibitors enhance in vitro susceptibility to chloroquine in resistant Plasmodium falciparum.

Chloroquine resistance in Plasmodium falciparum was reversed in vitro by the neuronal monoamine reuptake inhibitors and antidepressants desipramine, sertraline, fluoxetine, and norfluoxetine but not by carbamazepine, an antiseizure and mood-stabilizing tricyclic drug resembling desipramine which only weakly inhibits neuronal monoamine reuptake. These findings have important clinical implications for drug combination therapy.

1-Naphthylamine↗
PubMed1993

Transfected Leishmania expressing biologically active IFN-gamma.

Infection of susceptible BALB/c mice with Leishmania major leads to progressive infection with the failure to expand and activate Th1 CD4+ T cells that elaborate IFN-gamma, a critically implicated cytokine for control of disease. We used the recently described capacity to express foreign genes in trypanosomatids to introduce into Leishmania the murine IFN-gamma gene on a drug-selectable plasmid under the constitutive control of intergenic tubulin sequences. Several clones of L. major were established and demonstrated to contain IFN-gamma DNA and IFN-gamma RNA that was appropriately trans-spliced with the Leishmania-specific leader sequence, and to secrete IFN-gamma into the media. The secreted IFN-gamma was biologically active as assessed by up-regulation of class II MHC Ag and induction of macrophage nitric oxide synthase activity in a macrophage cell line. Infection of nude mice with IFN-gamma-containing organisms resulted in significantly slower progression of disease as compared to infection with organisms containing the empty plasmid, suggesting that biologically important activation of infected macrophages might be occurring in vivo. Infection of genetically susceptible BALB/c mice, however, did not impede the expansion of Th2 cells and the inexorable progression of disease. Despite the demonstration of increased levels of IFN-gamma transcription in vivo, induction of nitric oxide synthase in macrophages and expression of Ly-6, and IFN-gamma-inducible Ag, on CD4+ lymphocytes could not be shown. In all cases, organisms recovered from tissue amastigotes contained the IFN-gamma plasmid and secreted active IFN-gamma. The data confirm earlier studies that IFN-gamma alone is not sufficient to impede activation and maturation of Th2 cells in susceptible mice, even when targeted directly to the infected cell.

Animals↗
PubMed1993

Molecular characterization of the ldmdr1 multidrug resistance gene from Leishmania donovani.

The ldmdr1 gene that confers resistance to multiple structurally dissimilar hydrophobic drugs in Leishmania donovani has been isolated within a 5.4-kb XmaI fragment from a genomic library of L. donovani DNA and its protein coding region sequenced. The longest open reading frame within ldmdr1 encodes a 146.5-kDa protein of 1341 amino acids, designated LDMDR1. The primary structure and predicted membrane topology of LDMDR1 indicates that it is a member of the P-glycoprotein superfamily with the greatest homology to the mammalian multidrug resistance P-glycoproteins. A 2.3-kb SalI fragment derived from a second ldmdr1 allele was also cloned from the L. donovani library. Nucleotide sequence analysis of a portion of the SalI insert revealed 5 single base differences from its counterpart within the 5.4-kb XmaI fragment, one of which created a PvuI restriction site polymorphism. Southern blots of PvuI-digested DNA divulged that the amplified ldmdr1 gene copies in a multidrug-resistant L. donovani strain were all derived from the single ldmdr1 allele whose protein coding segment was sequenced in its entirety.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Mutational analysis of a signal sequence required for protein secretion in Leishmania major.

In eukaryotes, amino-terminal extensions, signal sequences, mediate the translocation of lysosomal, membrane and secreted proteins into the lumen of the endoplasmic reticulum (ER). Structure/function studies indicate that eukaryotic signal sequences are composed of 3 distinct domains, a positively charged amino-terminal domain, a central hydrophobic domain, and a polar carboxy-terminal domain. In an attempt to better understand protein trafficking in Leishmania we have constructed strains of Leishmania major that secrete an exogenous protein, interferon-gamma (IFN-gamma), under the control of a mammalian signal sequence. In this report we present a mutational analysis of this signal sequence. Deletion of the entire signal sequence or the hydrophobic core region prevents secretion of IFN-gamma and results in cytoplasmic expression of the protein. Mutations in the amino-terminal domain indicate that a net positive charge is not required for efficient secretion of IFN-gamma. Mutations in the carboxy-terminal domain are more complex and display two phenotypes, either they prevent expression of IFN-gamma or they have no effect on protein secretion. These results indicate that the function of the signal sequence in targeting proteins to the ER in Leishmania is similar to that observed in yeasts and higher eukaryotes and suggests that the Leishmania protein secretory apparatus may also be similar.

Amino Acid Sequence↗
PubMed1993

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii.

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5-30 times the IC50 (30 micrograms ml-1) of parental cells. The vinblastine-resistant parasites were also resistant to puromycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug efflux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdr1, and demonstrate that this gene is amplified on an extrachromosomal circle of 35-40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr1 and 83% identity with the L. donovani ldmdr1 gene. The lemdr1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.

Amino Acid Sequence↗
PubMed1993

Characterization of the pfmdr2 gene for Plasmodium falciparum.

We report the characterization of the pfmdr2 gene which is a gene related to the P-glycoprotein family but with a somewhat different structure than the mdr genes. Based on DNA sequence analysis of genomic clones, we have discovered that the pfmdr2 gene has 10 predicted transmembrane domains and a single ATP-binding site. In a homology search using GenBank sequences, we discovered that the pfmdr2 gene has a significant homology with the hmt1 gene in yeast. The yeast hmt1 gene is involved in cadmium resistance and is hypothesized to transport cadmium containing complexes from the cell. We have further characterized the pfmdr2 gene expression by northern analysis and discovered that it is expressed in a stage-specific manner, only at the trophozoite stage and not in ring stages. We have prepared a rabbit antibody to a recombinant fusion protein expressing a portion of the pfmdr2 coding region. In IFA analysis, this antibody stains trophozoites and not ring stages. Western analysis reveals a protein of approximately 110 kDa which is consistent with the size of the predicted open reading frame based on DNA sequence analysis. Based on this analysis and previous work, there is no evidence for a change in pfmdr2 expression in drug-resistant versus drug-sensitive parasites.

ATP-Binding Cassette Transporters↗