28 results on '"Keithly, Janet S."'
Search Results
2. Divergent polyamine metabolism in the Apicomplexa
- Author
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Cook, Tuesday, Roos, David, Morada, Mary, Zhu, Guan, Keithly, Janet S., Feagin, Jean E., Wu, Gang, and Yarlett, Nigel
- Subjects
Polyamines -- Research ,Biosynthesis -- Research ,Toxoplasma -- Research ,Molecular biology -- Research ,Biological sciences - Abstract
The lead enzymes of polyamine biosynthesis, i.e. ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), were not detected in Toxoplasma gondii [the limit of detection for ODC and ADC was 5 pmol [min.sup.-1] [(mg protein).sup.-1]], indicating that T. gondii lacks a forward-directed polyamine biosynthetic pathway, and is therefore a polyamine auxotroph. The biochemical results were supported by results obtained from data-mining the T. gondii genome. However, it was possible to demonstrate the presence of a highly active backconversion pathway that formed spermidine from spermine, and putrescine from spermidine, via the combined action of spermidine/ spermine [N.sup.1]-acetyltransferase (SSAT) or spermidine [N.sup.1]-acetyltransferase (SAT) and polyamine oxidase (PAO). With spermine as the substrate, T. gondii SSAT had a specific activity of 1.84 nmol [min.sup.-1] [(mg protein).sup.-1], and an apparent [K.sub.m] for spermine of 180 mM; with spermidine as the substrate, the SAT had a specific activity of 3.95 nmol [min.sup.-1] [(mg protein).sup.-1], and a [K.sub.m] for spermidine of 240 mM. T. gondii PAO had a specific activity of 10.6 nmol [min.sup.-1] [(mg protein).sup.-1], and a [K.sub.m] for acetylspermine of 36 mM. Furthermore, the results demonstrated that T. gondii SSAT was 50 % inhibited by 30 mM di(ethyl)norspermine. The parasite actively transported arginine and ornithine, which were converted via the arginine dihydrolase pathway to citrulline and carbamoyl phosphate, resulting in the formation of ATP via carbamate kinase. The lack of polyamine biosynthesis by T. gondii is contrasted with polyamine metabolism by other apicomplexans.
- Published
- 2007
3. Cryptosporidium parvum Cpn60 targets a relict organelle
- Author
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Riordan, Christina E., Ault, Jeffrey G., Langreth, Susan G., and Keithly, Janet S.
- Published
- 2003
- Full Text
- View/download PDF
4. Mitochondrial-type iron-sulfur cluster biosynthesis genes (IscS and IscU)in the apicomplexan Cryptosporidium parvum
- Author
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LaGier, Michael J., Tachezy, Jan, Stejskal, Frantisek, Kutisova, Katerina, and Keithly, Janet S.
- Subjects
Bacterial proteins -- Genetic aspects ,Bacterial proteins -- Physiological aspects ,Biosynthesis -- Analysis ,Brewer's yeast -- Genetic aspects ,Brewer's yeast -- Physiological aspects ,Chloroplasts -- Genetic aspects ,Chloroplasts -- Physiological aspects ,Coccidia -- Genetic aspects ,Coccidia -- Physiological aspects ,HIV (Viruses) -- Causes of ,Iron compounds -- Environmental aspects ,Iron compounds -- Physiological aspects ,Microbiology -- Research ,Mitochondria -- Genetic aspects ,Mitochondria -- Physiological aspects ,Phosphates -- Physiological aspects ,Sulfur -- Environmental aspects ,Sulfur -- Physiological aspects ,Biological sciences - Abstract
Several reports have indicated that the iron-sulfur [Fe-S] assemblly machinery in most eukaryotes is confined to the mitochondria and chloroplasts. The best-characterised and most highly conserved [Fe-S] assembly proteins are a pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS), and IscU, a protein functioning as a scaffold for the assembly of [Fe-S] prior to their incorporation into apoproteins. In this work, genes encoding IscS and IscU homologues have been isolated and characterized from the apicomplexan parasite Cryptosporidium parvum, an opportunistic pathogen in AIDS patients, for which no effective treatment is available. Primary sequence analysis (CplscS and CplscU) and phylogenetic studies (CplscS) indicate that both genes are most closely related to mitochondrial homologues from other organisms. Moreover, the N-terminal signal sequences of CplscS and CplscU predicted in silico specifically target green fluorescent protein to the mitochondrial network of the yeast Saccharomyces cerevisiae. Overall, these findings suggest that the previously identified mitochondrial relict of C. parvum may have been retained by the parasite as an intracellular site for [Fe-S] assembly.
- Published
- 2003
5. Cryptosporidiosis
- Author
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Chen, Xian-Ming, Keithly, Janet S., Paya, Carlos V., and LaRusso, Nicholas F.
- Subjects
Cryptosporidiosis -- Analysis - Abstract
The symptoms, diagnosis, treatment, and prevention of cryptosporidiosis are reviewed. This is a gastrointestinal infection with a parasite called Cryptosporidium. It causes diarrhea in healthy people, but can be much more serious in AIDS patients and other people with weakened immune systems.
- Published
- 2002
6. Cryptosporidium parvum appears to lack a plastid genome
- Author
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Zhu, Guan, Marchewska, Mary J., and Keithly, Janet S.
- Subjects
Microbiological research -- Analysis ,Plastids -- Research ,Genomes -- Research ,Biological sciences - Abstract
Research has been conducted on the plastid genome. Results indicate that no genome has been found in Cryptosporidium parvum.
- Published
- 2000
7. Evaluation of in vitro and in vivo activity of benzindazole-4,9-quinones against Cryptosporidium parvum
- Author
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Kayser, Oliver, Waters, W. Ray, Woods, Keith M., Upton, Steve J., Keithly, Janet S., Laatsch, Hartmut, and Kiderlen, Albrecht F.
- Published
- 2002
8. Current Concepts: Cryptosporidiosis
- Author
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Chen, Xian-Ming, Keithly, Janet S., Paya, Carlos V., and LaRusso, Nicholas F.
- Published
- 2002
9. Characterisation of a novel transporter from Cryptosporidiumparvum
- Author
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LaGier, Michael J., Keithly, Janet S., and Zhu, Guan
- Published
- 2002
- Full Text
- View/download PDF
10. Characterization of S-adenosylhomocysteine hydrolase from Cryptosporidium parvum.
- Author
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Čtrnáct, Vlasta, Stejskal, František, Keithly, Janet S., and Hrd, Ivan
- Subjects
HYDROLASES ,CRYPTOSPORIDIUM parvum ,APICOMPLEXA ,GENES ,PROTEINS ,AMINO acids ,POLYMERASE chain reaction ,CARRIER proteins - Abstract
The S-adenosylhomocysteine hydrolase from the apicomplexan Cryptosporidium parvum ( CpSAHH) has been characterized. CpSAHH is a single-copy, intronless gene of 1479 bp encoding a protein of 493 amino acids with a molecular mass of 55.6 kDa. Reverse transcriptase-polymerase chain reaction analysis confirmed that CpSAHH is expressed both in intracellular stages (in C. parvum-infected HCT-8 cells 24 h after infection) and in sporozoites. CpSAHH was expressed in Escherichia coli TB1 cells as a fusion with maltose-binding protein. The recombinant fusion was cleaved by Factor Xa and the enzymatic activity of both the fusion protein and the purified separated CpSAHH was measured. The enzymatic activity of CpSAHH was inhibited byd-eritadenine, S-DHPA and Ara-A. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
11. Localization of Pyruvate:NADP+ Oxidoreductase in Sporozoites of Cryptosporidium parvum.
- Author
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Ctrnacta, Vlasta, Ault, Jeffrey G., Stejskal, Frantisek, and Keithly, Janet S.
- Subjects
CRYPTOSPORIDIUM parvum ,IMMUNOFLUORESCENCE ,PYRUVATES ,FERREDOXIN-NADP reductase ,TRANSMISSION electron microscopy ,EUGLENA gracilis - Abstract
Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP
+ oxidoreductase ( CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body. [ABSTRACT FROM AUTHOR]- Published
- 2006
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- View/download PDF
12. Electron Tomographic and Ultrastructural Analysis of the Cryptosporidium parvum Relict Mitochondrion, its Associated Membranes, and Organelles.
- Author
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Keithly, Janet S., Langreth, Susan G., Buttle, Karolyn F., and Mannella, Carmen A.
- Subjects
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CRYPTOSPORIDIUM parvum , *COCCIDIA , *PROTOZOA , *ULTRASTRUCTURE (Biology) , *RELICTS (Biology) , *MITOCHONDRIA , *TRANSMISSION electron microscopy , *TOMOGRAPHY - Abstract
Reports on an ultrastructural analysis of the relict mitochondrion of Cryptosporidium parvum sporozoites that includes electron tomographic reconstruction. Presence of either a single, highly-folded inner membrane or multiple internal subcompartments; Lack of tubular "crista junctions" in the infoldings of the inner membrane; Suggestion that the retention of a relict mitochondrion is a strategy for compartmentalizing away from the cytosol toxic ferrous iron and sulfide.
- Published
- 2005
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13. A Narf-like gene from Cryptosporidium parvum resembles homologues observed in aerobic protists and higher eukaryotes
- Author
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Stejskal, František, Šlapeta, Jan, Čtrnáctá, Vlasta, and Keithly, Janet S.
- Subjects
APICOMPLEXA ,GENES ,CRYPTOSPORIDIUM parvum ,PHYLOGENY - Abstract
Here we report a Narf-like gene from the apicomplexan Cryptosporidium parvum (CpNARF). CpNARF is an intronless, single-copy gene of 1680 bp which encodes a putative protein of 560 amino acids with a calculated molecular mass of 63.1 kDa. This gene contains a single highly conserved N-terminal iron–sulfur cluster ([4Fe–4S]) binding site, as well as most of the H-cluster conserved residues. Reverse transcription polymerase chain reaction analysis indicates that CpNARF is expressed by the intracellular stages of C. parvum. Although the function of this gene is as yet unknown, phylogenetic analyses suggest that CpNARF belongs to the group of NARF-like proteins from aerobic protists and higher eukaryotes, which are thought to have had an ancestor in common with [Fe]-hydrogenases. [Copyright &y& Elsevier]
- Published
- 2003
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14. Characterization of S-adenosylmethionine synthetase in Cryptosporidium parvum (Apicomplexa)
- Author
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Šlapeta, Jan, Stejskal, František, and Keithly, Janet S.
- Subjects
CRYPTOSPORIDIUM parvum ,AMINO acids ,DIARRHEA - Abstract
The S-adenosylmethionine synthetase gene of the apicomplexan Cryptosporidium parvum (CpSAMS), an agent of diarrhea in immunocompromised and healthy humans and animals is described. CpSAMS is a single-copy, intronless gene of 1221 bp encoding a polypeptide of 406 amino acids with a molecular mass of 44.8 kDa. The gene is AT-rich (61.8%). CpSAMS was expressed in Escherichia coli TB1 cells as a fusion with maltose binding protein. The activity of the recombinant fusion was assayed, and was found to be inhibited by the methionine analog cycloleucine. In order to determine whether CpSAMS was differentially expressed during the life cycle of C. parvum, HCT-8 cells were infected with C. parvum and assayed over 72 h. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) confirmed the differential expression of CpSAMS. [Copyright &y& Elsevier]
- Published
- 2003
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15. α-Proteobacterial Relationship of Apicomplexan Lactate and Malate Dehydrogenases.
- Author
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ZHU, GUAN and KEITHLY, JANET S.
- Published
- 2002
- Full Text
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16. Pyruvate : NADP Oxidoreductase from the Mitochondrion of Euglena gracilis and from the Apicomplexan Cryptosporidium parvum: A Biochemical Relic Linking Pyruvate Metabolism in Mitochondriate and Amitochondriate Protists.
- Author
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Rotte, Carmen, Stejskal, Frantisek, Zhu, Guan, Keithly, Janet S., and Martin, William
- Abstract
Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O2-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O2-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO2 in air and with 2% CO2 in N2. The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes. [ABSTRACT FROM PUBLISHER]
- Published
- 2001
- Full Text
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17. Ornithine Decarboxylase and Trypanothione Reductase Genes in Leishmania braziliensis guyanensis.
- Author
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KEITHLY, JANET S.
- Published
- 1989
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18. Introduction.
- Author
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KEITHLY, JANET S.
- Subjects
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PROTISTA , *UNICELLULAR organisms - Abstract
The article discusses issues published within the issue, including facets of acidocalcisomes and protists by Roberto Docampo and protein trafficking by Marilyn Parsons.
- Published
- 2009
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19. Minimizing DNA recombination during long RT-PCR
- Author
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Fang, Guowei, Zhu, Guan, Burger, Harold, Keithly, Janet S, and Weiser, Barbara
- Published
- 1998
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20. Expression and functional characterization of a giant Type I fatty acid synthase (CpFAS1) gene from Cryptosporidium parvum
- Author
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Zhu, Guan, Li, Yanan, Cai, Xiaomin, Millership, Jason J., Marchewka, Mary J., and Keithly, Janet S.
- Subjects
- *
CRYPTOSPORIDIOSIS , *MALTOSE , *CARRIER proteins , *FATTY acids - Abstract
A 25-kb CpFAS1 gene from Cryptosporidium parvum has been engineered and expressed as five individual maltose-binding protein (MBP)-fusion proteins: an N-terminal loading unit, three fatty acyl elongation modules, and a C-terminal reductase. Enzymatic activities of all domains (except the reductase) were individually assayed as recombinant proteins. The preferred substrate for the fatty acyl ligase (AL) domain in the loading unit was palmitic acid (C16:0). However, a competition assay suggests that the AL domain could also utilize other fatty acids as substrates (i.e., C12:0–C24:0), albeit with reduced activity. Among the three elongation modules, enzymatic activities were detected for ketoacyl synthase (KS), acyl transferase (AT), dehydrase (DH), enoyl reductase (ER), and ketoacyl reductase (KR) domains, which suggests that these modules were involved in the elongation of a saturated fatty acyl chain that would be C6 longer (e.g., C22:0) than the precursor (e.g., C16:0). In addition, the KS activity could be specifically inhibited by cerulenin (
IC50∼1.5 μM), reinforcing the notion that CpFAS1 could be exploited as potential drug target. Since C. parvum lacks other fatty acid synthases, these observations imply that this parasite may not be capable of synthesizing fatty acids de novo. [Copyright &y& Elsevier]- Published
- 2004
- Full Text
- View/download PDF
21. Mitochondrial-type iron-sulfur cluster biosynthesis genes (lscS and lscU) in the apicomplexan Cryptosporidium parvum.
- Author
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LaGier, Michael J., Tachezy, Jan, Stejskal, Frantisek, Kutisova, Katerina, and Keithly, Janet S.
- Subjects
- *
CRYPTOSPORIDIUM parvum , *EUKARYOTIC cells , *MITOCHONDRIA , *PATHOGENIC microorganisms , *BIOSYNTHESIS , *MICROBIOLOGY - Abstract
Several reports have indicated that the iron-sulfur cluster [Fe-S] assembly machinery in most eukaryotes is confined to the mitochondria and chloroplasts. The best-characterized and most highly conserved [Fe-S] assembly proteins are a pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS), and IscU, a protein functioning as a scaffold for the assembly of [Fe-S] prior to their incorporation into apoproteins. In this work, genes encoding IscS and IscU homologues have been isolated and characterized from the apicomplexan parasite Cryptosporidium parvum, an opportunistic pathogen in AIDS patients, for which no effective treatment is available. Primary sequence analysis (CplscS and CplscU) and phytogenetic studies (CplscS) indicate that both genes are most closely related to mitochondrial homologues from other organisms. Moreover, the N-terminal signal sequences of CplscS and CplscU predicted in silico specifically target green fluorescent protein to the mitochondrial network of the yeast Saccharomyces cerevisiae. Overall, these findings suggest that the previously identified mitochondrial relict of C. parvum may have been retained by the parasite as an intracellular site for [Fe-S] assembly. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
- View/download PDF
22. Cryptosporidium parvum: the first protist known to encode a putative polyketide synthase
- Author
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Zhu, Guan, LaGier, Michael J., Stejskal, Frantisek, Millership, Jason J., Cai, Xiaomin, and Keithly, Janet S.
- Subjects
- *
CRYPTOSPORIDIUM parvum , *POLYKETIDES , *ACYLTRANSFERASES , *ANTISENSE DNA , *POLYMERASE chain reaction - Abstract
We are reporting a putative multifunctional Type I polyketide synthase (PKS) gene from the apicomplexan Cryptosporidium parvum (CpPKS1). The 40 kb intronless open reading frame (ORF) predicts a single polypeptide of 13,414 amino acids with a molecular mass of 1516.5 kDa. Sequence analysis identified at least 29 enzymatic domains within this protein. These domains are organized into an N-terminal loading unit, seven polyketide chain elongation modules, and a carboxy terminator unit. The loading domain consists of an acyl-CoA ligase (AL) and an acyl carrier protein (ACP). All seven elongation modules contain between two and five of the six domains required for the elongation of two-carbon (C2) acyl units, i.e. ketoacyl synthase, acyl transferase, dehydrase, enoyl reductase, ketoreductase and/or ACP. The carboxy terminator is homologous to various reductases, suggesting that the final elongated product is not hydrolytically released by thioesterases as observed in most Type I PKS and all fatty acid synthetase (FAS) systems, but by a reducing reaction, which has been demonstrated in some non-ribosomal peptide synthase systems. The protein sequence and domain organization of CpPKS1 protein resembles a previously reported C. parvum fatty acid synthase (CpFAS1), which is encoded by a 25 kb ORF. Maximum likelihood phylogenetic analysis of acyl transferases within PKS/FAS from C. parvum and other organisms clearly differentiates acetate-extending clades from those incorporating propionate. All acyl transferase domains from CpPKS1, and a previously reported CpFAS1, clustered within the acetate-extending group, suggesting the likelihood that only non-methylated C2 units are incorporated by C. parvum polyketide and fatty acid synthases. The expression of CpPKS1 was confirmed by reverse transcription-polymerase chain reaction and immunofluorescence microscopy. Many polyketides are medically significant antibiotics, anticancer agents, toxins, or signaling molecules. Therefore, it is interesting to speculate what role CpPKS1 might play in this apicomplexan and the disease caused by this opportunistic infection of AIDS patients. [Copyright &y& Elsevier]
- Published
- 2002
- Full Text
- View/download PDF
23. A new real-time PCR assay for improved detection of the parasite Babesia microti.
- Author
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Teal AE, Habura A, Ennis J, Keithly JS, and Madison-Antenucci S
- Subjects
- Babesia microti genetics, DNA, Protozoan genetics, DNA, Ribosomal genetics, Humans, Parasitemia diagnosis, Parasitemia parasitology, RNA, Ribosomal, 18S genetics, Sensitivity and Specificity, United States, Babesia microti isolation & purification, Babesiosis diagnosis, Babesiosis parasitology, Molecular Diagnostic Techniques methods, Real-Time Polymerase Chain Reaction methods
- Abstract
Babesiosis is an emerging zoonosis with important public health implications, as the incidence of the disease has risen dramatically over the past decade. Because the current gold standard for detection of Babesia is microscopic examination of blood smears, accurate identification requires trained personnel. Species in the genus cannot be distinguished microscopically, and Babesia can also be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. To allow more accurate diagnosis in a format that is accessible to a wider variety of laboratories, we developed a real-time PCR assay targeting the 18S rRNA gene of Babesia microti, the dominant babesiosis pathogen in the United States. The real-time PCR is performed on DNA extracted from whole-blood specimens and detects Babesia microti with a limit of detection of ∼100 gene copies in 5 μl of blood. The real-time PCR assay was shown to be 100% specific when tested against a panel of 24 organisms consisting of Babesia microti, other Babesia species, Plasmodium species, tick-borne and other pathogenic bacteria, and other blood-borne parasites. The results using clinical specimens show that the assay can detect infections of lower parasitemia than can be detected by microscopic examination. This method is therefore a rapid, sensitive, and accurate method for detection of Babesia microti in patient specimens.
- Published
- 2012
- Full Text
- View/download PDF
24. Cellular compartmentalization: Protists do it their way. Introduction.
- Author
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Keithly JS
- Subjects
- Animals, Eukaryota genetics, Eukaryota metabolism, Evolution, Molecular, Metabolic Networks and Pathways, Organelles metabolism, Eukaryota physiology, Organelles physiology
- Published
- 2009
- Full Text
- View/download PDF
25. Characterization of S-adenosylhomocysteine hydrolase from Cryptosporidium parvum.
- Author
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Ctrnáctá V, Stejskal F, Keithly JS, and Hrdý I
- Subjects
- Adenine analogs & derivatives, Adenine pharmacology, Adenosylhomocysteinase antagonists & inhibitors, Adenosylhomocysteinase genetics, Amino Acid Sequence, Animals, Cryptosporidium parvum genetics, Escherichia coli genetics, Gene Expression, Gene Expression Regulation, Developmental, Introns, Molecular Sequence Data, Molecular Weight, RNA, Messenger biosynthesis, RNA, Messenger genetics, RNA, Protozoan biosynthesis, RNA, Protozoan genetics, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Vidarabine pharmacology, Adenosylhomocysteinase chemistry, Adenosylhomocysteinase metabolism, Cryptosporidium parvum enzymology
- Abstract
The S-adenosylhomocysteine hydrolase from the apicomplexan Cryptosporidium parvum (CpSAHH) has been characterized. CpSAHH is a single-copy, intronless gene of 1479 bp encoding a protein of 493 amino acids with a molecular mass of 55.6 kDa. Reverse transcriptase-polymerase chain reaction analysis confirmed that CpSAHH is expressed both in intracellular stages (in C. parvum-infected HCT-8 cells 24 h after infection) and in sporozoites. CpSAHH was expressed in Escherichia coli TB1 cells as a fusion with maltose-binding protein. The recombinant fusion was cleaved by Factor Xa and the enzymatic activity of both the fusion protein and the purified separated CpSAHH was measured. The enzymatic activity of CpSAHH was inhibited by d-eritadenine, S-DHPA and Ara-A.
- Published
- 2007
- Full Text
- View/download PDF
26. Localization of pyruvate:NADP+ oxidoreductase in sporozoites of Cryptosporidium parvum.
- Author
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Ctrnacta V, Ault JG, Stejskal F, and Keithly JS
- Subjects
- Animals, Blotting, Western, Cryptosporidium parvum cytology, Cryptosporidium parvum genetics, Cryptosporidium parvum growth & development, Cytosol enzymology, Euglena gracilis cytology, Euglena gracilis enzymology, Ketone Oxidoreductases genetics, Ketone Oxidoreductases immunology, Microscopy, Confocal, Microscopy, Electron, Transmission, Microscopy, Fluorescence, NADPH-Ferrihemoprotein Reductase genetics, NADPH-Ferrihemoprotein Reductase immunology, Organelles enzymology, Protozoan Proteins analysis, Pyruvate Synthase genetics, Pyruvate Synthase immunology, Sporozoites cytology, Sporozoites genetics, Cryptosporidium parvum enzymology, Ketone Oxidoreductases analysis, NADPH-Ferrihemoprotein Reductase analysis, Pyruvate Synthase analysis, Sporozoites enzymology
- Abstract
Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body.
- Published
- 2006
- Full Text
- View/download PDF
27. Cryptosporidium parvum mitochondrial-type HSP70 targets homologous and heterologous mitochondria.
- Author
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Slapeta J and Keithly JS
- Subjects
- Amino Acid Sequence, Animals, Cryptosporidium parvum genetics, Cryptosporidium parvum ultrastructure, Escherichia coli Proteins genetics, Gene Expression, HSP70 Heat-Shock Proteins chemistry, HSP70 Heat-Shock Proteins genetics, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Mitochondria genetics, Mitochondria ultrastructure, Molecular Sequence Data, Phylogeny, Protein Sorting Signals, Protein Structure, Secondary, Protein Transport, Sequence Alignment, Sporozoites ultrastructure, Cryptosporidium parvum metabolism, HSP70 Heat-Shock Proteins metabolism, Mitochondria metabolism
- Abstract
A mitochondrial HSP70 gene (Cp-mtHSP70) is described for the apicomplexan Cryptosporidium parvum, an agent of diarrhea in humans and animals. Mitochondrial HSP70 is known to have been acquired from the proto-mitochondrial endosymbiont. The amino acid sequence of Cp-mtHSP70 shares common domains with mitochondrial and proteobacterial homologues, including 34 amino acids of an NH2-terminal mitochondrion-like targeting presequence. Phylogenetic reconstruction places Cp-mtHSP70 within the mitochondrial clade of HSP70 homologues. Using reverse transcription-PCR, Cp-mtHSP70 mRNA was observed in C. parvum intracellular stages cultured in HCT-8 cells. Polyclonal antibodies to Cp-mtHSP70 recognize a approximately 70-kDa protein in Western blot analysis of sporozoite extracts. Both fluorescein- and immunogold-labeled anti-Cp-mtHSP70 localize to a single mitochondrial compartment in close apposition to the nucleus. Furthermore, the NH2-terminal presequence of Cp-mtHSP70 can correctly target green fluorescent protein to the single mitochondrion of the apicomplexan Toxoplasma gondii and the mitochondrial network of the yeast Saccharomyces cerevisiae. When this presequence was truncated, the predicted amphiphilic alpha-helix was shown to be essential for import into the yeast mitochondrion. These data further support the presence of a secondarily reduced relict mitochondrion in C. parvum.
- Published
- 2004
- Full Text
- View/download PDF
28. Alpha-proteobacterial relationship of apicomplexan lactate and malate dehydrogenases.
- Author
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Zhu G and Keithly JS
- Subjects
- Alphaproteobacteria enzymology, Amino Acid Sequence, Animals, Base Sequence, Cryptosporidium parvum genetics, DNA, Mitochondrial chemistry, DNA, Mitochondrial genetics, DNA, Protozoan chemistry, DNA, Protozoan genetics, Gammaproteobacteria genetics, L-Lactate Dehydrogenase chemistry, Malate Dehydrogenase chemistry, Molecular Sequence Data, Phylogeny, Sequence Alignment, Sequence Analysis, DNA, Alphaproteobacteria genetics, Cryptosporidium parvum enzymology, L-Lactate Dehydrogenase genetics, Malate Dehydrogenase genetics
- Abstract
We have cloned and sequenced a lactate dehydrogenase (LDH) gene from Cryptosporidium parvum (CpLDH1). With this addition, and that of four recently deposited alpha-proteobacterial malate dehydrogenase (MDH) genes, the phylogenetic relationships among apicomplexan LDH and bacterial MDH were re-examined. Consistent with previous studies, our maximum likelihood (ML) analysis using the quartet-puzzling method divided 105 LDH/MDH enzymes into five clades, and confirmed that mitochondrial MDH is a sister clade to those of y-proteobacteria, rather than to alpha-proteobacteria. In addition, a Cryptosporidium parvum MDH (CpMDH1) was identified from the ongoing Cryptosporidium genome project that appears to belong to a distinct clade (III) comprised of 22 sequences from one archaebacterium, numerous eubacteria, and several apicomplexans. Using the ML puzzling test and bootstrapping analysis with protein distance and parsimony methods, the resulting trees not only robustly confirmed the alpha-proteobacterial relationship of apicomplexan LDH/MDH, but also supported a monophyletic relationship of CpLDH1 with CpMDHI. These data suggest that, unlike most other eukaryotes, the Apicomplexa may be one of the few lineages retaining an alpha-proteobacterial-type MDH that could have been acquired from an ancestral alpha-proteobacterium through primary endosymbiosis giving rise to the mitochondria, or through an unknown lateral gene transfer (LGT) event.
- Published
- 2002
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