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1.
Parasitology ; 148(9): 1074-1082, 2021 08.
Article in English | MEDLINE | ID: mdl-33966667

ABSTRACT

Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.


Subject(s)
Acanthamoeba castellanii/drug effects , Amebicides/pharmacology , Genes, Protozoan/drug effects , Plant Extracts/pharmacology , Senna Plant/chemistry , Transcription, Genetic/drug effects , Acanthamoeba castellanii/genetics , Plant Extracts/chemistry
2.
Malar J ; 19(1): 238, 2020 Jul 06.
Article in English | MEDLINE | ID: mdl-32631416

ABSTRACT

BACKGROUND: Since 2014, seasonal malaria chemoprevention (SMC) with amodiaquine-sulfadoxine-pyrimethamine (AQ-SP) has been implemented on a large scale during the high malaria transmission season in Burkina Faso. This paper reports the prevalence of microscopic and submicroscopic malaria infection at the outset and after the first round of SMC in children under 5 years old in Bama, Burkina Faso, as well as host and parasite factors involved in mediating the efficacy and tolerability of SMC. METHODS: Two sequential cross-sectional surveys were conducted in late July and August 2017 during the first month of SMC in a rural area in southwest Burkina Faso. Blood smears and dried blood spots were collected from 106 to 93 children under five, respectively, at the start of SMC and again 3 weeks later. Malaria infection was detected by microscopy and by PCR from dried blood spots. For all children, day 7 plasma concentrations of desethylamodiaquine (DEAQ) were measured and CYP2C8 genetic variants influencing AQ metabolism were genotyped. Samples were additionally genotyped for pfcrt K76T and pfmdr1 N86Y, molecular markers associated with reduced amodiaquine susceptibility. RESULTS: 2.8% (3/106) of children were positive for Plasmodium falciparum infection by microscopy and 13.2% (14/106) by nested PCR within 2 days of SMC administration. Three weeks after SMC administration, in the same households, 4.3% (4/93) of samples were positive by microscopy and 14.0% (13/93) by PCR (p = 0.0007). CYP2C8*2, associated with impaired amodiaquine metabolism, was common with an allelic frequency of 17.1% (95% CI 10.0-24.2). Day 7 concentration of DEAQ ranged from 0.48 to 362.80 ng/mL with a median concentration of 56.34 ng/mL. Pfmdr1 N86 predominated at both time points, whilst a non-significant trend towards a higher prevalence of pfcrt 76T was seen at week 3. CONCLUSION: This study showed a moderate prevalence of low-level malaria parasitaemia in children 3 weeks following SMC during the first month of administration. Day 7 concentrations of the active DEAQ metabolite varied widely, likely reflecting variability in adherence and possibly metabolism. These findings highlight factors that may contribute to the effectiveness of SMC in children in a high transmission setting.


Subject(s)
Amodiaquine/analogs & derivatives , Antimalarials/blood , Cytochrome P-450 CYP2C8/genetics , Drug Resistance/genetics , Genes, Protozoan/drug effects , Malaria, Falciparum/prevention & control , Polymorphism, Genetic/drug effects , Amodiaquine/blood , Amodiaquine/therapeutic use , Antimalarials/therapeutic use , Burkina Faso/epidemiology , Chemoprevention , Child, Preschool , Cross-Sectional Studies , Female , Humans , Infant , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Male , Plasma/chemistry
3.
Nat Commun ; 10(1): 371, 2019 01 22.
Article in English | MEDLINE | ID: mdl-30670687

ABSTRACT

Studies of Plasmodium vivax gene expression are complicated by the lack of in vitro culture system and the difficulties associated with studying clinical infections that often contain multiple clones and a mixture of parasite stages. Here, we characterize the transcriptomes of P. vivax parasites from 26 malaria patients. We show that most parasite mRNAs derive from trophozoites and that the asynchronicity of P. vivax infections is therefore unlikely to confound gene expression studies. Analyses of gametocyte genes reveal two distinct clusters of co-regulated genes, suggesting that male and female gametocytes are independently regulated. Finally, we analyze gene expression changes induced by chloroquine and show that this antimalarial drug efficiently eliminates most P. vivax parasite stages but, in contrast to P. falciparum, does not affect trophozoites.


Subject(s)
Chloroquine/pharmacology , Gene Expression Regulation/drug effects , Plasmodium vivax/drug effects , Plasmodium vivax/genetics , Transcriptome/drug effects , Antimalarials/pharmacology , Cell Cycle/drug effects , Cell Cycle/genetics , Female , Genes, Protozoan/drug effects , Genes, Protozoan/genetics , Genome, Protozoan/drug effects , Genome, Protozoan/genetics , Humans , Malaria/drug therapy , Malaria, Falciparum/drug therapy , Malaria, Vivax/drug therapy , Male , Multigene Family , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Plasmodium vivax/pathogenicity , RNA, Messenger/metabolism , Trophozoites/drug effects , Trophozoites/genetics
4.
J Pineal Res ; 66(2): e12484, 2019 03.
Article in English | MEDLINE | ID: mdl-29480948

ABSTRACT

Malaria causes millions of deaths worldwide and is considered a huge burden to underdeveloped countries. The number of cases with resistance to all antimalarials is continuously increasing, making the identification of novel drugs a very urgent necessity. A potentially very interesting target for novel therapeutic intervention is the parasite mitochondrion. In this work, we studied in Plasmodium falciparum 3 genes coding for proteins homologues of the mammalian FIS1 (Mitochondrial Fission Protein 1) and DRP1 (Dynamin Related Protein 1) involved in mitochondrial fission. We studied the expression of P. falciparum genes that show ample sequence and structural homologies with the mammalian counterparts, namely FIS1, DYN1, and DYN2. The encoded proteins are characterized by a distinct pattern of expression throughout the erythrocytic cycle of P. falciparum, and their mRNAs are modulated by treating the parasite with the host hormone melatonin. We have previously reported that the knockout of the Plasmodium gene that codes for protein kinase 7 is essential for melatonin sensing. We here show that PfPk7 knockout results in major alterations of mitochondrial fission genes expression when compared to wild-type parasites, and no change in fission proteins expression upon treatment with the host hormone. Finally, we have compared the morphological characteristics (using MitoTracker Red CMX Ros) and oxygen consumption properties of P. falciparum mitochondria in wild-type parasites and PfPk7 Knockout strains. A novel GFP construct targeted to the mitochondrial matrix to wild-type parasites was also developed to visualize P. falciparum mitochondria. We here show that, the functional characteristics of P. falciparum are profoundly altered in cells lacking protein kinase 7, suggesting that this enzyme plays a major role in the control of mitochondrial morphogenesis and maturation during the intra-erythrocyte cell cycle progression.


Subject(s)
Genes, Protozoan/drug effects , Melatonin/pharmacology , Mitochondrial Dynamics/drug effects , Mitochondrial Dynamics/physiology , Plasmodium falciparum/metabolism , Dynamins/metabolism , Erythrocytes/parasitology , Gene Knockout Techniques , Green Fluorescent Proteins , Humans , Mitochondria/drug effects , Mitochondrial Proteins/metabolism , Plasmodium falciparum/drug effects , Protein Kinases/metabolism
5.
Article in English | MEDLINE | ID: mdl-29238701

ABSTRACT

Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position -170 to -111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (-151 to -136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Emetine/pharmacology , Entamoeba histolytica/drug effects , Entamoeba histolytica/metabolism , Heat-Shock Response/physiology , Protozoan Proteins/genetics , Transcriptional Activation/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Base Sequence , Binding Sites , Drug Resistance, Multiple/genetics , Electroporation , Entamoeba histolytica/genetics , Entamoeba histolytica/growth & development , Gene Expression Regulation/drug effects , Genes, Protozoan/drug effects , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/physiology , Protozoan Proteins/analysis , Protozoan Proteins/physiology , Transcription Factors/metabolism , Transcription, Genetic , Transfection , Trophozoites/drug effects
6.
Malar J ; 16(1): 238, 2017 06 06.
Article in English | MEDLINE | ID: mdl-28587615

ABSTRACT

BACKGROUND: The current trend of Plasmodium vivax cases imported from Southeast Asia into China has sharply increased recently, especially from the China-Myanmar border (CMB) area. High recombination rates of P. vivax populations associated with varied transmission intensity might cause distinct local selective pressures. The information on the genetic variability of P. vivax in this area is scant. Hence, this study assessed the genetic diversity of P. vivax genome sequence in CMB area and aimed to provide information on the positive selection of new gene loci. RESULTS: This study reports a genome-wide survey of P. vivax in CMB area, using blood samples from local patients to identify population-specific selective processes. The result showed that considerable genetic diversity and mean pair-wise divergence among the sequenced P. vivax isolates were higher in some important gene families. Using the standardized integrated haplotype score (|iHS|) for all SNPs in chromosomal regions with SNPs above the top 1% distribution, it was observed that the top score locus involved 356 genes and most of them are associated with red blood cell invasion and immune evasion. The XP-EHH test was also applied and some important genes associated with anti-malarial drug resistance were observed in high positive scores list. This result suggests that P. vivax in CMB area is facing more pressure to survive than any other region and this has led to the strong positive selection of genes that are associated with host-parasite interactions. CONCLUSIONS: This study suggests that greater genetic diversity in P. vivax from CMB area and positive selection signals in invasion and drug resistance genes are consistent with the history of drug use during malaria elimination programme in CMB area. Furthermore, this result also demonstrates that haplotype-based detecting selection can assist the genome-wide methods to identify the determinants of P. vivax diversity.


Subject(s)
Antimalarials/pharmacology , Drug Resistance , Genome, Protozoan , Plasmodium vivax/drug effects , Plasmodium vivax/genetics , China , Genes, Protozoan/drug effects , Genetic Variation , Malaria, Vivax/prevention & control
7.
BMC Genomics ; 9: 388, 2008 Aug 18.
Article in English | MEDLINE | ID: mdl-18706115

ABSTRACT

BACKGROUND: Translation of the genome sequence of Plasmodium sp. into biologically relevant information relies on high through-put genomics technology which includes transcriptome analysis. However, few studies to date have used this powerful approach to explore transcriptome alterations of P. falciparum parasites exposed to antimalarial drugs. RESULTS: The rapid action of artesunate allowed us to study dynamic changes of the parasite transcriptome in synchronous parasite cultures exposed to the drug for 90 minutes and 3 hours. Developmentally regulated genes were filtered out, leaving 398 genes which presented altered transcript levels reflecting drug-exposure. Few genes related to metabolic pathways, most encoded chaperones, transporters, kinases, Zn-finger proteins, transcription activating proteins, proteins involved in proteasome degradation, in oxidative stress and in cell cycle regulation. A positive bias was observed for over-expressed genes presenting a subtelomeric location, allelic polymorphism and encoding proteins with potential export sequences, which often belonged to subtelomeric multi-gene families. This pointed to the mobilization of processes shaping the interface between the parasite and its environment. In parallel, pathways were engaged which could lead to parasite death, such as interference with purine/pyrimidine metabolism, the mitochondrial electron transport chain, proteasome-dependent protein degradation or the integrity of the food vacuole. CONCLUSION: The high proportion of over-expressed genes encoding proteins exported from the parasite highlight the importance of extra-parasitic compartments as fields for exploration in drug research which, to date, has mostly focused on the parasite itself rather than on its intra and extra erythrocytic environment. Further work is needed to clarify which transcriptome alterations observed reflect a specific response to overcome artesunate toxicity or more general perturbations on the path to cellular death.


Subject(s)
Antimalarials/pharmacology , Artemisinins/pharmacology , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Analysis of Variance , Animals , Artesunate , Cells, Cultured , Gene Expression Profiling , Gene Expression Regulation/drug effects , Genes, Protozoan/drug effects , Life Cycle Stages , Oligonucleotide Array Sequence Analysis , Plasmodium falciparum/growth & development , RNA, Protozoan/genetics , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
8.
BMC Genomics ; 8: 216, 2007 Jul 05.
Article in English | MEDLINE | ID: mdl-17612405

ABSTRACT

BACKGROUND: Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known. RESULTS: In order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)). CONCLUSION: This work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite.


Subject(s)
Entamoeba histolytica/drug effects , Entamoeba histolytica/genetics , Gene Expression Regulation/drug effects , Genes, Protozoan/drug effects , Histones/metabolism , Hydroxamic Acids/pharmacology , Animals , Butyric Acid/pharmacology , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Propionates/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sodium Acetate/pharmacology
9.
BMC Genomics ; 8: 7, 2007 Jan 05.
Article in English | MEDLINE | ID: mdl-17207281

ABSTRACT

BACKGROUND: In higher eukaryotes DNA methylation regulates important biological functions including silencing of gene expression and protection from adverse effects of retrotransposons. In the protozoan parasite Entamoeba histolytica, a DNA methyltransferase has been identified and treatment with 5-azacytidine (5-AzaC), a potent inhibitor of DNA methyltransferase, has been reported to attenuate parasite virulence. However, the overall extent of DNA methylation and its subsequent effects on global gene expression in this parasite are currently unknown. RESULTS: In order to identify the genome-wide effects of DNA methylation in E. histolytica, we used a short oligonucleotide microarray representing 9,435 genes (approximately 95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 microM 5-AzaC for up to one week. Overall, 2.1% of genes tested were transcriptionally modulated under these conditions. 68 genes were upregulated and 131 genes down regulated (2-fold change; p-value < 0.05). Sodium-bisulfite treatment and sequencing of genes indicated that there were at least two subsets of genes with genomic DNA methylation in E. histolytica: (i) genes that were endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induced transcriptional de-repression, and (ii) genes that have genomic DNA methylation, but which were not endogenously silenced by the methylation. We identified among the genes down regulated by 5-AzaC treatment a cysteine proteinase (2.m00545) and lysozyme (52.m00148) both of which have known roles in amebic pathogenesis. Decreased expression of these genes in the 5-AzaC treated E. histolytica may account in part for the parasites reduced cytolytic abilities. CONCLUSION: This work represents the first genome-wide analysis of DNA-methylation in Entamoeba histolytica and indicates that DNA methylation has relatively limited effects on gene expression in this parasite.


Subject(s)
Azacitidine/pharmacology , Entamoeba histolytica/drug effects , Entamoeba histolytica/growth & development , Gene Expression Regulation/drug effects , Genes, Protozoan/physiology , Protozoan Proteins/biosynthesis , Animals , CHO Cells , Cricetinae , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA Methylation , Entamoeba histolytica/pathogenicity , Gene Silencing , Genes, Protozoan/drug effects , Genome, Protozoan/physiology , Mesocricetus , Microarray Analysis , Protozoan Proteins/antagonists & inhibitors , Virulence/drug effects
10.
Aquat Toxicol ; 81(1): 99-105, 2007 Feb 15.
Article in English | MEDLINE | ID: mdl-17188762

ABSTRACT

Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T. thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system.


Subject(s)
Gene Expression/drug effects , Tetrahymena thermophila/drug effects , Trialkyltin Compounds/toxicity , Water Pollutants, Chemical/toxicity , Animals , Base Sequence , DNA Primers/chemistry , Expressed Sequence Tags/chemistry , Genes, Protozoan/drug effects , Molecular Sequence Data , Polymerase Chain Reaction/veterinary , Tetrahymena thermophila/genetics
11.
Biol Pharm Bull ; 26(8): 1174-7, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12913271

ABSTRACT

The cellular slime mold Dictyostelium discoideum expresses three genes (sodA, sodB and sodC) encoding the extracellular Cu/Zn superoxide dismutases. Following H(2)O(2) treatment, the expression of sodA and sodB increased while that of sodC decreased. The sodC null strain formed multinucleate cells in a shaking culture. These results suggest that sodC plays a unique role in Dictyostelium discoideum.


Subject(s)
Dictyostelium/genetics , Dictyostelium/metabolism , Escherichia coli Proteins , Genes, Protozoan , Giant Cells/enzymology , Superoxide Dismutase/deficiency , Superoxide Dismutase/genetics , Amino Acid Sequence , Animals , Base Sequence , Dictyostelium/cytology , Genes, Protozoan/drug effects , Giant Cells/cytology , Giant Cells/drug effects , Hydrogen Peroxide/pharmacology , Molecular Sequence Data , Mutagenesis, Insertional , Sequence Analysis , Superoxide Dismutase/biosynthesis
12.
Bull Exp Biol Med ; 134(4): 379-81, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12533765

ABSTRACT

Microscopic examination does not allow differentiation of drug-resistant P. falciparum infection relapse from reinfection. However, this differential diagnosis is essential for adequate therapy. Three highly polymorphic P. falciparum genes (msp1, msp2, and glurp) and their alleles reflecting the structural state of these genes were used as genetic markers for differential diagnosis by PCR with internal primers. In 27 patients the characteristics of these alleles were identical before treatment with artersunate and during repeated manifestation of symptoms 14-28 days after the end of therapy, which attested to malaria relapses. In 24 patients the structure of these allele before mefloquine therapy and during repeated manifestation of the symptoms after 2-3 months was different, which attested to reinfection.


Subject(s)
DNA, Protozoan/genetics , Malaria, Falciparum/diagnosis , Plasmodium falciparum/genetics , Polymerase Chain Reaction/methods , Alleles , Animals , Antimalarials/therapeutic use , Artemisinins/therapeutic use , Artesunate , Diagnosis, Differential , Drug Resistance, Multiple , Genes, Protozoan/drug effects , Genetic Markers , Humans , Mefloquine/therapeutic use , Polymorphism, Genetic , Recurrence , Sesquiterpenes/therapeutic use , Time Factors
13.
Mol Biochem Parasitol ; 107(1): 57-69, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10717302

ABSTRACT

During the construction of a physical map for Leishmania major (LV39) chromosome 2 we have rescued and characterized a L. major (LV39) derived genomic clone bearing solely as insert a long stretch of the miniexon gene array. The recombinant was devised as a tool to study the effect of miniexon overexpression on virulence and growth advantage. Such clone, 32D05, contains approximately 40 kb of the miniexon tandem array. We have examined the course of infection in susceptible BALB/c mice inoculated with transfectants carrying 32D05 as an episome. The study was carried out in two different clonal lines of L. major: virulent line LV39 (clone 5) and avirulent LT252 (CC1 clone). The results presented here indicate that high levels of miniexon expression affect negatively the ability of once virulent lines to induce lesions when injected in susceptible mice.


Subject(s)
Exons/genetics , Gene Expression Regulation , Genes, Protozoan , Leishmania major/genetics , Leishmania major/pathogenicity , Animals , Anthelmintics/pharmacology , Clone Cells , Exons/drug effects , Female , Gene Dosage , Gene Expression Regulation/drug effects , Genes, Protozoan/drug effects , Genome, Protozoan , Hygromycin B/therapeutic use , Leishmania major/drug effects , Leishmaniasis, Cutaneous/drug therapy , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Cutaneous/parasitology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , RNA, Protozoan/analysis , Transfection
15.
Biochim Biophys Acta ; 1446(3): 273-85, 1999 Sep 03.
Article in English | MEDLINE | ID: mdl-10524202

ABSTRACT

The effects of distamycin A on Acanthamoeba transcription, growth and differentiation were determined. Distamycin A inhibits transcription both in vitro and in vivo and can displace from DNA the transcription activator TATA binding protein promoter binding factor (TPBF). Inhibition in vivo is surprisingly selective for large rRNA precursors, 5S rRNA, profilin, S-adenosylmethionine synthetase, and extendin. Transcription from the TATA binding protein (TBP), TPBF, protein disulfide isomerase, tubulin and RNA polymerase II large subunit genes is only slightly inhibited. Moreover the rate of 5S rRNA transcription eventually recovers and exceeds that of untreated cells, while profilin transcription remains inhibited. Distamycin A inhibition is accompanied by a complex pattern of alterations to steady state levels of mRNAs. Actin, profilin and S-adenosylmethionine synthetase mRNAs are degraded, whereas mRNA encoding TBP is increased slightly in abundance. Transcription inhibition is accompanied by cessation of growth and severe morphological changes to Acanthamoeba, which are consistent with loss of production of mRNA encoding cytoskeletal proteins. Distamycin A also prevents starvation-induced differentiation of Acanthamoeba, in part due to complete prevention of cellulose production and cell wall formation.


Subject(s)
Acanthamoeba/drug effects , Antiprotozoal Agents/pharmacology , Distamycins/pharmacology , Genes, Protozoan/drug effects , RNA, Protozoan/biosynthesis , Acanthamoeba/genetics , Acanthamoeba/growth & development , Animals , Cellulose/metabolism , Cytoskeletal Proteins/genetics , DNA-Binding Proteins/genetics , Protein Binding/drug effects , RNA, Messenger/metabolism , TATA-Box Binding Protein , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription, Genetic/drug effects
16.
Dev Genet ; 23(3): 230-46, 1998.
Article in English | MEDLINE | ID: mdl-9842717

ABSTRACT

The promoter of the Dictyostelium glycogen phosphorylase-2 (gp2) gene possesses a profound AT-bias, typical of promoters in this organism. To understand how Dictyostelium achieves specificity during transcriptional regulation under the constraint of this highly biased nucleotide composition, we have documented the changes in chromatin structure associated with developmental induction of gp2 gene expression. DNase I hypersensitive analyses indicated the presence of several developmentally regulated nuclease-sensitive sites located upstream of the start codon: two strong sites at approximately -250 bp and -350 bp and three substantially weaker sites at -290 bp, -445 bp, and -505 bp. In vitro footprint analyses using nuclear extracts derived from several stages of development (corresponding to varying levels of gp2 expression) revealed three large regions of occupation that were developmentally regulated and corresponded to these nuclease-sensitive sites: -227 to -294 bp (domain 1), -327 to -383 bp (domain 2), and -416 to -534 bp (domain 3). The presence and the extent of the three regulatory domains was confirmed by in vivo footprint analyses spanning the same developmental time points. Southwestern analyses using probes encompassing these footprints demonstrated that probes corresponding to domains 1 and 3 both interacted with 83 and 77 kDa peptides. The domain 3 probe also interacted with a 92 kDa peptide, while only a 62 kDa peptide is recognized by the domain 2 probe. In all cases, peptides capable of binding these probes were found in nuclear extracts derived from differentiated cells and not in undifferentiated cell nuclear extract. Using nuclear extract from differentiated cells and probes corresponding to the three domains, gel mobility shift analyses detected ladders of retarded bands for both domains 1 and 3 and three major retarded bands for domain 2. These results suggest that specificity in transcriptional activation in the AT-rich promoters of Dictyostelium may be achieved by requiring multiple protein-DNA and/or protein-protein interactions to occur before induction can proceed.


Subject(s)
Dictyostelium/enzymology , Dictyostelium/genetics , Genes, Protozoan , Phosphorylases/genetics , Animals , Base Composition , Binding Sites/genetics , Cyclic AMP/pharmacology , DNA Footprinting , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , DNA-Binding Proteins/metabolism , Deoxyribonuclease I , Dictyostelium/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Genes, Protozoan/drug effects , Promoter Regions, Genetic , Protozoan Proteins/metabolism , Transcription, Genetic
17.
FEMS Microbiol Lett ; 140(2-3): 121-4, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-8764472

ABSTRACT

The untranslatable, RNA polymerase II-dependent gene (dutA) of Dictyostelium discoideum is induced early in development. However, unlike other early genes, dutA induction was not affected by cAMP pulses and occurred normally in various cAMP-related mutant cells, the results indicating that this induction depended solely on factors other than cAMP. In the knockout strain of the catalytic subunit of protein kinase A, dutA expression was severely blocked and not recovered by cAMP pulses. This demonstrates that even the cAMP-independent gene, dutA, requires protein kinase A for its expression.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Dictyostelium/genetics , Dictyostelium/metabolism , Genes, Fungal , Genes, Protozoan , Animals , Cyclic AMP/pharmacology , Cyclic AMP-Dependent Protein Kinases/genetics , Dictyostelium/drug effects , Gene Deletion , Gene Expression/drug effects , Gene Targeting , Genes, Fungal/drug effects , Genes, Protozoan/drug effects , RNA Polymerase II/metabolism
18.
Nucleic Acids Res ; 23(13): 2526-30, 1995 Jul 11.
Article in English | MEDLINE | ID: mdl-7630731

ABSTRACT

Both prokaryotic and eukaryotic cells contain multiple forms of ribonuclease H, a ribonuclease that specifically degrades the RNA strand of RNA-DNA hybrids and which has been implicated in the processing of initiator RNAs and in the removal of RNA primers from Okazaki fragments. The Crithidia fasciculata RNH1 gene encodes an RNase H and was shown to be a single-copy gene in this diploid trypanosomatid. The RNH1 gene has been disrupted by targeted gene disruption using hygromycin or G418 drug-resistance cassettes. Major active forms of RNase H (38 and 45 kDa) were observed on activity gels of extracts of wild-type cells or cells in which one allele of RNH1 was disrupted. Both the 38 and 45 kDa activities were absent in extracts of cells in which both alleles of RNH1 were disrupted indicating that both forms of the C.fasciculata RNase H are encoded by the RNH1 gene.


Subject(s)
Cinnamates , Crithidia fasciculata/genetics , Genes, Protozoan , Ribonuclease H/genetics , Animals , Blotting, Southern , DNA Restriction Enzymes , Drug Resistance/genetics , Gene Transfer Techniques , Genes, Protozoan/drug effects , Hygromycin B/analogs & derivatives , Hygromycin B/pharmacology , Kanamycin Kinase , Neomycin , Phosphotransferases (Alcohol Group Acceptor)/genetics
20.
Parasitol Res ; 81(3): 207-16, 1995.
Article in English | MEDLINE | ID: mdl-7770426

ABSTRACT

Ultrastructural evidence is presented for the presence of plastid-like organelles in Toxoplasma gondii, Sarcocystis muris, Babesia ovis, and Plasmodium falciparum. In addition, it was shown that merozoites of T. gondii contain protochlorophyllidae a and traces of chlorophyll a bound to the photosynthetic reaction centers I PS I and PS II. A psbA gene was isolated from merozoites of S. muris by the polymerase chain reaction (PCR). Partial sequencing of the PCR product revealed that the herbicide-binding region is highly conserved. Therefore, it is likely that the sensitivity of apicomplexans to the herbicide toltrazuril depends on the interaction of the herbicide with the D1 protein of the photosynthetic reaction center of the parasite's organelles.


Subject(s)
Apicomplexa/chemistry , Photosynthesis , Triazines , Amino Acid Sequence , Animals , Apicomplexa/drug effects , Apicomplexa/genetics , Apicomplexa/ultrastructure , Base Sequence , Chlorophyll/analysis , Chlorophyll A , Conserved Sequence , DNA, Protozoan/analysis , Extrachromosomal Inheritance , Genes, Protozoan/drug effects , Genes, Protozoan/genetics , Humans , Light-Harvesting Protein Complexes , Mice , Mice, Inbred BALB C , Mice, Inbred Strains , Molecular Sequence Data , Organelles/ultrastructure , Photosynthesis/drug effects , Photosynthetic Reaction Center Complex Proteins/drug effects , Photosynthetic Reaction Center Complex Proteins/ultrastructure , Photosystem II Protein Complex , Phylogeny , Protochlorophyllide/analysis , Sarcocystis/genetics , Sequence Alignment , Toxoplasma/genetics , Toxoplasma/physiology , Triazines/therapeutic use
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