Biomedical subjects

Jennifer M Fitzpatrick

Publications and source records attributed to Jennifer M Fitzpatrick.

7 recordsLinked to original sources

PubMed2006

Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases.

Egg production is responsible for life cycle progression and host immunopathology during schistosomiasis, with the associated parasite molecules being investigated as potential novel chemotherapeutic targets. Here, we characterize two Schistosoma mansoni products, tyrosinase 1 and tyrosinase 2 (SmTYR1/SmTYR2) and show that their diphenol oxidase enzyme activities are critical for eggshell formation and production. The genes encoding these bifunctional enzymes (monophenol and diphenol oxidases) result from a duplication event that likely occurred before speciation and exist in the parasite's genome as multiple copies, which are linked and localized to chromosomes 4 and W. SmTYR1/SmTYR2 transcription and diphenol oxidase action are developmentally regulated with most enzyme activity localized to the eggshell-producing cells contained within the vitellaria of adult female worms. Importantly, kojic-acid mediated inhibition (IC50=0.5 microM) of SmTYR1/SmTYR2's diphenol oxidase activity during in vitro culture of sexually mature adult worms resulted in a significant decrease in the production of phenotypically normal eggs. Therefore our data suggest that SmTYR1/2 inhibition represents a novel and potentially effective strategy for combating schistosomiasis and furthermore, it may point to new methods for combinatorial control of immunopathology and egg transmission during platyhelminth infection.

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PubMed2006

Dioecious Schistosoma mansoni express divergent gene repertoires regulated by pairing.

Pairing of adult Schistosoma mansoni parasites initiates a cascade of events including mating and egg production that ultimately leads to immuno-pathological lesions during schistosomiasis. To identify genes associated with this important biological process, we studied parasites isolated from single- versus mixed-sex cercariae-infected mice using DNA microarray analysis to uncover pair-regulated transcriptional profiles. We report that: (i) transcriptomes of parasites isolated from single-sex infections are significantly more complex than their mixed-sex counterparts; (ii) transcriptomes of single-sex males are distinct from mixed-sex males; and (iii) not all transcripts, previously hypothesized to be critical in female egg production, are regulated by pairing.

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PubMed2006

Gender-specific expression of complex-type N-glycans in schistosomes.

Sex-specific gene expression by Schistosoma mansoni worms has been demonstrated at the transcriptome as well as the proteome levels. In view of the important role of glycans in the biology of schistosomes and the interaction with their human host, we have investigated the sex-specific protein glycosylation. Mass spectrometric profiling and structural characterization of PNGase F-released N-glycans revealed the following gender-specific glycosylation patterns: Complex-type N-glycans of females mainly carried Gal(beta1-4)GlcNAc (LacNAc) and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc (Lewis x) antennae structures, whereas GalNAc(beta1-4)GlcNAc- (LacdiNAc; LDN) and GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc (LDN-F) were prevalent in N-glycans from males. LDN(-F) motifs were found to occur as repeats on the antennae of large N-glycans that contained up to seven LDN(-F) units. The female complex-type glycans were mostly di-antennary and tri-antennary, whereas male structures were predominantly of the mono-antennary and di-antennary type. Oligomannosidic N-glycans were expressed at similar levels in females and males. The localization of the sex-biased glycan motifs was studied by immunofluorescence microscopy using defined anti-glycan monoclonal antibodies (mAbs). The Lewis x element was strongly expressed in the gut of both males and females, but with respect to tegument localization, the females expressed this structure, while Lewis x seemed to be almost completely absent from the male tegument. The expression of LDN-F was predominantly detected in the parenchyma of both male and female worms as well as in the tegument of the male ventral cavity facing the female. LDN was detected in the tegument of male and female worms at similar levels. The sex-specific expression and differential localization of these antigenic glycan motifs in schistosomes may play a role in male-female interactions during conjugal biology and may lead to a differential immune reaction of the host to the two sexes.

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PubMed2006

Schistosoma mansoni: DNA microarray gene expression profiling during the miracidium-to-mother sporocyst transformation.

For the human blood fluke, Schistosoma mansoni, the developmental period that constitutes the transition from miracidium to sporocyst within the molluscan host involves major alterations in morphology and physiology. Although the genetic basis for this transformation process is not well understood, it is likely to be accompanied by changes in gene expression. In an effort to reveal genes involved in this process, we performed a DNA microarray analysis of expressed mRNAs between miracidial and 4 d old in vitro-cultured mother sporocyst stages of S. mansoni. Fluorescently labeled, dsDNA targets were synthesized from miracidia and sporocyst total RNA and hybridized to oligonucleotide DNA microarrays containing 7335 S. mansoni sequences. Fluorescence intensity ratios were statistically compared between five biologically replicated experiments to identify particular transcripts that displayed stage-associated expression within miracidial and sporocyst mRNA populations. A total of 361 sequences showed stage-associated expression in miracidia, while 273 probes displayed sporocyst-associated expression. Differentially expressed mRNAs were annotated with gene ontology terminology based on BLAST homology using high throughput gene ontology functional annotation toolkit (HT-GO-FAT) and clustered using the GOblet GO browser software. A subset of genes displaying stage-associated expression by microarray analyses was verified utilizing real-time quantitative PCR. The use of DNA microarrays for the profiling of gene expression in early-developing S. mansoni larvae provides a starting point for expanding our understanding of the genes that may be involved in the establishment of parasitism and maintenance of infection in these important life cycle stages.

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PubMed2005

An oligonucleotide microarray for transcriptome analysis of Schistosoma mansoni and its application/use to investigate gender-associated gene expression.

Global profiling transcriptomes of parasitic helminths offers the potential to simultaneously identify co-ordinately expressed genes, novel genetic programs and uniquely utilized metabolic pathways, which together provide an extensive and new resource for vaccine and drug discovery. We have exploited this post-genomic approach to fabricate the first oligonucleotide DNA microarray for gene expression analysis of the parasitic trematode Schistosoma mansoni. A total of 17,329 S. mansoni DNA sequences were used to design a microarray consisting of 7335 parasite elements or approximately 50% of this parasite's transcriptome. Here, we describe the design of this new microarray resource and its evaluation by extending studies into gender-associated gene expression in adult schistosomes. We demonstrate a high degree of reproducibility in detecting transcriptional differences among biologically replicated experiments and the ability of the microarray to distinguish between the expression of closely related gene family members. Importantly, for issues related to sexual dimorphism, labour division, gamete production and drug target discovery, 197 transcripts demonstrated a gender-biased pattern of gene expression in the adult schistosome, greatly extending the number of sex-associated genes. These data demonstrate the power of this new resource to facilitate a greater understanding into the biological complexities of schistosome development and maturation useful for identifying novel intervention strategies.

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PubMed2004

Gender-associated gene expression in two related strains of Schistosoma japonicum.

Host inflammatory responses directed against eggs laid by sexually-mature Schistosoma japonicum female worms instigate lesion formation and associated clinical pathologies during infection. To identify parasite gene transcripts that associate with egg production and to characterise sexually-mature adult gene expression profiles of two related Chinese strains, S. japonicum cDNA microarrays were fabricated using 457 ESTs originating from three parasite developmental stages. Twenty-two female-associated and 8 male-associated gene transcripts were identified in the adult Anhui strain whereas 21 female-associated and 7 male-associated gene transcripts were revealed in the adult Zhejiang strain. RT-PCR analysis, in situ enzyme localisation studies and enzymatic assays confirmed the cDNA microarray results, and importantly, provided information previously unappreciated in schistosome conjugal biology. Specifically, our novel findings include the female-specific expression of genes putatively involved in haemoglobin digestion and eggshell formation including extracellular superoxide dismutase, two histidine-rich proteins, a large blood-brain barrier amino acid transporter and two tyrosinase orthologues. In contrast, transcripts involved in mechanical support (actin), cytoskeletal infrastructure (e.g. dynein light chain 3 and myosin regulatory light chain) and tegumental biology (e.g. TM4SF and Sj25) were more highly represented in adult male schistosomes. Together these data establish a transcriptional basis for adult schistosome labour division and expands the list of novel S. japonicum gender-associated gene transcripts that may be considered targets for improved intervention strategies.

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PubMed2004

Gene expression studies using self-fabricated parasite cDNA microarrays.

DNA microarray platforms represent a functional genomics technology that uses structured information obtained from genomic sequencing efforts as a means to study transcriptional processes in a systematic and high-throughput manner. Specifically in this chapter, we outline the ordered processes involved in large-scale parasite gene expression studies including complementary (cDNA) microarray fabrication, total RNA isolation, cDNA labeling using fluorochromes, and DNA:DNA hybridization. Methods described herein were adapted for the study of schistosome sexual maturation and developmental biology but could be easily modified for the study of any additional parasitological system.

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