John Dame | University of Florida (original) (raw)

Papers by John Dame

International Journal for Parasitology, Dec 1, 1997

Accurate snail intermediate host infection prevalence data have the potential to be extremely use... more Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of > 99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.

Veterinary Parasitology, Feb 1, 2001

Veterinary Parasitology, Feb 1, 1993

Most genetic work to distinguish strains of parasitic helminths focuses on searching for genetic ... more Most genetic work to distinguish strains of parasitic helminths focuses on searching for genetic markers to correlate with phenotypes of interest, but in this study genetic diversity among individual Ostertagia ostertagi adults is partitioned into components within and between populations. Restriction fragment polymorphism data on mitochondrial DNA from ten individual worms from each of five different parasite populations are analyzed. Three of these populations are characterized by arrested larval development (hypobiosis) over the summer months, and the other two by hypobiosis over the winter months. Sequence divergence is scored by the presence or absence of 37 different restriction sites. Although the populations are genetically differentiated with respect to the timing of hypobiosis, greater than 98% of the total mitochondrial DNA sequence diversity is partitioned within a single population, and the geographic distribution of individual mitochondrial DNA haplotypes suggests high gene flow among populations. Further, estimates of within-population mitochondrial DNA diversity are five to ten times greater in O. ostertagi than typical estimates reported for species in other taxa.

Journal of Parasitology, Dec 1, 1995

Equine protozoal myeloencephalitis (EPM) is a neurologic disease of horses caused by Sarcocystis ... more Equine protozoal myeloencephalitis (EPM) is a neurologic disease of horses caused by Sarcocystis neurona. The horse is a dead-end host for S. neurona and the definitive and intermediate hosts have not previously been identified. We hypothesized that S. neurona is actually Sarcocystis falcatula, a parasite that cycles in nature between Virginia opossums (Didelphis virginiana) and any of a variety of avian intermediate hosts. We extracted DNA from S. falcatula sarcocysts in the muscle of a brown-headed cowbird (Molothrus ater) and from schizonts in a fixed specimen of lung from a Moluccan cockatoo (Cacatua moluccensis). Three segments of the small subunit ribosomal RNA (SSURNA) gene, containing a total of 742 nucleotides, were amplified by the polymerase chain reaction, sequenced, and compared with the SSURNA sequence from two isolates of S. neurona. The S. falcatula sequence was identical to the sequence of the S. neurona isolate UCD-1 and differed in only 3 positions from isolate SN5. Recent evidence, also based on SSURNA sequences, implicates the opossum as the definitive host of S. neurona. Based on the SSURNA gene sequences S. falcatula and S. neurona are synonymous, thus the parasite cycles between opossums and birds maintaining a reservoir of the organism from which horses can be infected.

Journal of Parasitology, Dec 1, 1997

This is a report of the isolation and in vitro propagation of merozoites of Sarcocystis falcatula... more This is a report of the isolation and in vitro propagation of merozoites of Sarcocystis falcatula. Culture-derived S. falcatula merozoites remained infectious after several passages in tissue culture as determined by the susceptibility of experimentally inoculated Melopsittacus undulatus that died of pulmonary sarcocystosis after experimental inoculation. There was no obvious clinical disease or lesion when Sarcocystis neurona merozoites were used to inoculate M. undulatus. On the basis of the biological infectivity data, S. falcatula and S. neurona are not synonymous as suggested from limited molecular data.

Journal of Biological Chemistry, Mar 1, 2010

European journal of biochemistry, Feb 1, 1984

The two large ribsomal RNA subunits of Schistosoma mansoni are encoded within a 10 000-base seque... more The two large ribsomal RNA subunits of Schistosoma mansoni are encoded within a 10 000-base sequence, which is tandemly repeated in the schistosome genome. Restriction endonuclease digestion with Bam HI cuts the rRNA gene into three fragments, which have been clones separately in pBR322 and used to constract a physical map of the gene. The sequence encoding the smaller rRNA subunit is about 2000 bases in length and is situated on the 5' sie of the sequence encoding the larger subunit, which is about 4000 bases. Approximately 4000 bases of the rRNA gene are spacer and do not code for mature rRNA. There are approximately 100 copies of the rRNA gene per haploid genome of which about 10% exhibit length heterogeneity, as judged by the hybridization of the rDNA plasmids to restriction endonuclease digests of genomic DNA. These structural variants appear to contain additional DNA sequences at more than one site within the gene and are polymorphic within the species S. mansoni.

Science, Aug 24, 1984

Malaria parasites can be grouped evolutionarily by analysis of DNA composition and genome arrange... more Malaria parasites can be grouped evolutionarily by analysis of DNA composition and genome arrangement. Those that vary widely with regard to host range, morphology, and biological characteristics fit into only a small number of distinctive groups. The DNA of the human parasite Plasmodium falciparum fits into a group that includes rodent and avian malarias and is unlike the DNA of

The Journal of Infectious Diseases, Jul 1, 1985

Parasitology Today, Oct 1, 2000

Further analyses of the data, including cluster analysis and redundancy determination, transcript... more Further analyses of the data, including cluster analysis and redundancy determination, transcript reconstruction and protein prediction, are ongoing in collaboration with the South African National Bioinformatics Institute (http://www.sanbi.ac.za/malaria-genesearch/student.progress.html), and will be published shortly. In addition, all P. berghei clones, both as glycerol stocks of transformed E. coli and DNA minipreps, have been submitted to the Malaria Research and Reference Reagent Resource Center (MR4) [see J.H. Adams et al. Parasitol. Today 16, 89, 2000], a facility developed by NIAID for the distribution of reference malaria reagents (http://www.malaria.mr4.org/mr4pages/index.html). The P. vivax project has recently been completed and the complete set of 10 000 sequence tags and clones will also be submitted to MR4 shortly. As the data from the P. falciparum Genome Project are annotated and released, the sequence tags will be reanalyzed in an attempt to identify homologous genes. Having immediate access in a rodent malaria model to the counterpart of genes from P. falciparum will provide opportunities to test the function of those genes by genetic manipulation. In addition, access to the corresponding genes from P. vivax will aid in the development and design of vaccines and drugs active against both P. falciparum and P. vivax. These gene sequence tag projects represent a unique resource for the malaria community.

Molecular and Biochemical Parasitology, Aug 1, 2003

Experimental Parasitology, Feb 1, 2001

Journal of Molecular Biology, Jun 1, 2003

The malarial aspartic proteinases (plasmepsins) have been discovered in several species of Plasmo... more The malarial aspartic proteinases (plasmepsins) have been discovered in several species of Plasmodium, including all four of the human malarial pathogens. In P.falciparum, plasmepsins I, II, IV and HAP have been directly implicated in hemoglobin degradation during malaria infection, and are now considered targets for anti-malarial drug design. The plasmepsins are produced from inactive zymogens, proplasmepsins, having unusually long N-terminal prosegments of more than 120 amino acids. Structural and biochemical evidence suggests that the conversion process of proplasmepsins to plasmepsins differs substantially from the gastric and plant aspartic proteinases. Instead of blocking substrate access to a pre-formed active site, the prosegment enforces a conformation in which proplasmepsin cannot form a functional active site. We have determined crystal structures of plasmepsin and proplasmepsin from P.vivax. The three-dimensional structure of P.vivax plasmepsin is typical of the monomeric aspartic proteinases, and the structure of P.vivax proplasmepsin is similar to that of P.falciparum proplasmepsin II. A dramatic refolding of the mature N terminus and a large (18 degrees ) reorientation of the N-domain between P.vivax proplasmepsin and plasmepsin results in a severe distortion of the active site region of the zymogen relative to that of the mature enzyme. The present structures confirm that the mode of inactivation observed originally in P.falciparum proplasmepsin II, i.e. an incompletely formed active site, is a true structural feature and likely represents the general mode of inactivation of the related proplasmepsins.

Parasitology Research, Oct 1, 2000

Molecular and Biochemical Parasitology, Apr 1, 1994

A clone encoding the aspartic proteinase (PFAPD) from Plasmodium falciparum strain HB3 was obtain... more A clone encoding the aspartic proteinase (PFAPD) from Plasmodium falciparum strain HB3 was obtained during the course of a project designed to sequence and identify the protein coding regions of the parasite's genome. The protein encoded by the clone contains a sequence identical to the N-terminal sequence determined for an aspartic proteinase isolated from the digestive vacuole of P.falciparum and demonstrated to participate in the hemoglobin digestive pathway (D. Goldberg, personal communication). The translated polypeptide sequence encompasses a number of features characteristic of aspartic proteinases, having >30% identity and > 50% similarity overall to human cathepsin D, cathepsin E and renin. A model of the three-dimensional structure of PFAPD was constructed using rule-based procedures. This confirms that the primary sequence may be folded as a single chain into a three dimensional structure closely resembling those of other known aspartic proteinases. It includes a lengthy prosegment, two typical-hydrophobichydrophobic-Asp-Thr/Ser-Gly motifs and a tyrosine residue positioned in a fl-hairpin loop. The distribution of hydrophobic residues throughout the active site cleft is indicative of a likely preference for hydrophobic polypeptide substrates. The recombinant form of this enzyme expressed using the pGEX2T vector in Escherichia coli is active in digesting hemoglobin at acidic pH and in hydrolyzing a synthetic peptide corresponding to the putative initial cleavage site in hemoglobin. Activity is inhibited completely by pepstatin, confirming the identity of PFAPD as a member of the aspartic proteinase family. Specific mRNA for PFAPD is expressed in the erythrocytic stages of the life cycle.

Kluwer Academic Publishers eBooks, May 16, 2006

... Ben Dunn,1 Jennifer Westling,1 Mezeda Meze,1 Sheetal Nagar,1 Jeannette Gootjes,1 Patty Cipull... more ... Ben Dunn,1 Jennifer Westling,1 Mezeda Meze,1 Sheetal Nagar,1 Jeannette Gootjes,1 Patty Cipullo,1 Howard Saft,1 Amit Mathur,1 Tim Lee ... 8. Silva, MA, Lee, AY, Gulnik, SV, Majer, P., Collins, J., Bhat, TN, Collins, PJ, Cachau, RE, Luker, KE, Gluzman, IY, Francis, SE, Oksman, A ...

mSphere, Oct 28, 2020

The malaria parasite, Plasmodium falciparum, was introduced into Hispaniola and other regions of ... more The malaria parasite, Plasmodium falciparum, was introduced into Hispaniola and other regions of the Americas through the slave trade spanning the 16th through the 19th centuries. During this period, more than 12 million Africans were brought across the Atlantic to the Caribbean and other regions of the Americas. Since malaria is holoendemic in West Africa, a substantial percentage of these individuals carried the parasite. St. Domingue on Hispaniola, now modern-day Haiti, was a major port of disembarkation, and malaria is still actively transmitted there. We undertook a detailed study of the phylogenetics of the Haitian parasites and those from Colombia and Peru utilizing whole-genome sequencing. Principal-component and phylogenetic analyses, based upon single nucleotide polymorphisms (SNPs) in protein coding regions, indicate that, despite the potential for millions of introductions from Africa, the Haitian parasites share an ancestral relationship within a well-supported monophyletic clade with parasites from South America, while belonging to a distinct lineage. This result, in stark contrast to the historical record of parasite introductions, is best explained by a severe population bottleneck experienced by the parasites introduced into the Americas. Here, evidence is presented for targeted selection of rare African alleles in genes which are expressed in the mosquito stages of the parasite's life cycle. These genetic markers support the hypothesis that the severe population bottleneck was caused by the required adaptation of the parasite to transmission by new definitive hosts among the Anopheles (Nyssorhynchus) spp. found in the Caribbean and South America. IMPORTANCE Historical data suggest that millions of P. falciparum parasite lineages were introduced into the Americas during the transAtlantic slave trade, which would suggest a paraphyletic origin of the extant isolates in the Western Hemisphere. Our analyses of whole-genome variants show that the American parasites belong to a wellsupported monophyletic clade. We hypothesize that the required adaptation to American vectors created a severe bottleneck, reducing the effective introduction to a few lineages. In support of this hypothesis, we discovered genes expressed in the mosquito stages of the life cycle that have alleles with multiple, high-frequency or fixed, nonsynonymous mutations in the American populations which are rarely found in African isolates. These alleles appear to be in gene products critical for transmission through the anopheline vector. Thus, these results may inform efforts to develop novel transmissionblocking vaccines by identifying parasite proteins functionally interacting with the vector that are important for successful transmission. Further, to the best of our knowledge,

Molecular and Biochemical Parasitology, May 1, 2004

Plasmepsin 4 from Plasmodium falciparum and orthologs from Plasmodium malariae, Plasmodium ovale ... more Plasmepsin 4 from Plasmodium falciparum and orthologs from Plasmodium malariae, Plasmodium ovale and Plasmodium vivax have been expressed in recombinant form, and properties of the active site of each enzyme characterized by kinetic analysis. A panel of chromogenic peptide substrates systematically substituted at the P3, P2, P2 and P3 positions was used to estimate enzyme/ligand interactions in the corresponding enzyme subsites based upon kinetic data. The kinetic parameters k cat , K m and k cat /K m were measured to identify optimal substrates for each enzyme and also sequences that were readily cleaved by the plasmepsins but poorly by host aspartic peptidases. Computer generated models were utilized to compare enzyme structures and interpret kinetic results. The orthologous plasmepsins share highly similar subsite specificities. In the S3 and S2 subsites, the plasmepsin 4 orthologs all preferred hydrophobic amino acid residues, Phe or Ile, but rejected charged residues such as Lys or Asp. In S2 and S3 subsites, these plasmepsins tolerated both hydrophobic and hydrophilic residues. Subsite specificities of the plasmepsin 4 family of orthologs are similar to those of human cathepsins D and E, except in S3 where the plasmepsins accept substrates containing Ser significantly better than either of these human aspartic proteases. Peptidomimetic methyleneamino reduced-peptide inhibitors, which have inhibition constants in the picomolar range, were prepared for each plasmepsin 4 ortholog based upon substrate preferences. A peptidomimetic inhibitor designed for plasmepsin 4 from P. falciparum having Ser in P3 had the lowest K i of the series of inhibitors prepared, but did not significantly improve the selectivity of the inhibitor for plasmepsin 4 versus human cathepsin D.

Acta Crystallographica Section D-biological Crystallography, Feb 22, 2006