Biomedical subjects

Jay R Radke

Publications and source records attributed to Jay R Radke.

7 recordsLinked to original sources

PubMed2006

Changes in the expression of human cell division autoantigen-1 influence Toxoplasma gondii growth and development.

Toxoplasma is a significant opportunistic pathogen in AIDS, and bradyzoite differentiation is the critical step in the pathogenesis of chronic infection. Bradyzoite development has an apparent tropism for cells and tissues of the central nervous system, suggesting the need for a specific molecular environment in the host cell, but it is unknown whether this environment is parasite directed or the result of molecular features specific to the host cell itself. We have determined that a trisubstituted pyrrole acts directly on human and murine host cells to slow tachyzoite replication and induce bradyzoite-specific gene expression in type II and III strain parasites but not type I strains. New mRNA synthesis in the host cell was required and indicates that novel host transcripts encode signals that were able to induce parasite development. We have applied multivariate microarray analyses to identify and correlate host gene expression with specific parasite phenotypes. Human cell division autoantigen-1 (CDA1) was identified in this analysis, and small interfering RNA knockdown of this gene demonstrated that CDA1 expression causes the inhibition of parasite replication that leads subsequently to the induction of bradyzoite differentiation. Overexpression of CDA1 alone was able to slow parasite growth and induce the expression of bradyzoite-specific proteins, and thus these results demonstrate that changes in host cell transcription can directly influence the molecular environment to enable bradyzoite development. Investigation of host biochemical pathways with respect to variation in strain type response will help provide an understanding of the link(s) between the molecular environment in the host cell and parasite development.

Animals↗

PubMed2005

The transcriptome of Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown. RESULTS: We have used serial analysis of gene expression (SAGE) to define the Toxoplasma gondii transcriptome of the intermediate-host life cycle that leads to the formation of the bradyzoite/tissue cyst. A broad view of gene expression is provided by >4-fold coverage from nine distinct libraries (approximately 300,000 SAGE tags) representing key developmental transitions in primary parasite populations and in laboratory strains representing the three canonical genotypes. SAGE tags, and their corresponding mRNAs, were analyzed with respect to abundance, uniqueness, and antisense/sense polarity and chromosome distribution and developmental specificity. CONCLUSION: This study demonstrates that phenotypic transitions during parasite development were marked by unique stage-specific mRNAs that accounted for 18% of the total SAGE tags and varied from 1-5% of the tags in each developmental stage. We have also found that Toxoplasma mRNA pools have a unique parasite-specific composition with 1 in 5 transcripts encoding Apicomplexa-specific genes functioning in parasite invasion and transmission. Developmentally co-regulated genes were dispersed across all Toxoplasma chromosomes, as were tags representing each abundance class, and a variety of biochemical pathways indicating that trans-acting mechanisms likely control gene expression in this parasite. We observed distinct similarities in the specificity and expression levels of mRNAs in primary populations (Day-6 post-sporozoite infection) that occur prior to the onset of bradyzoite development that were uniquely shared with the virulent Type I-RH laboratory strain suggesting that development of RH may be arrested. By contrast, strains from Type II-Me49B7 and Type III-VEGmsj contain SAGE tags corresponding to bradyzoite genes, which suggests that priming of developmental expression likely plays a role in the greater capacity of these strains to complete bradyzoite development.

Animals↗

PubMed2004

Identification of a sporozoite-specific member of the Toxoplasma SAG superfamily via genetic complementation.

Toxoplasma gondii sporozoites possess an array of stage-specific antigens that are localized to the membrane and internal cellular space, as well as secreted into the primary parasitophorous vacuole. Specific labelling of viable sporozoites excysted from oocysts reveals a complex admixture of surface proteins partially shared with tachyzoites. SAG1, SRS3 and SAG3 were detected on sporozoites as well as numerous minor antigens. In contrast, tachyzoite SAG2A and B were completely absent whereas a dominant 25 kDa protein was unique to the sporozoite surface. The sporozoite gene encoding this protein was identified in tachyzoites genetically complemented with a sporozoite cDNA library and cloned via site-specific recombination into a bacterial shuttle vector. The sporozoite cDNA identified in these experiments encoded a protein with conserved structural features of the prototypical T. gondii SAG1 (P30) and shared sequence identity with surface proteins from Sarcocystis spp. This new member of the SAG superfamily was designated SporoSAG. Expression of SporoSAG in tachyzoites conferred enhanced invasion on transgenic parasites suggesting a role for this protein in oocyst/sporozoite transmission to susceptible hosts.

Amino Acid Sequence↗

PubMed2003

A change in the premitotic period of the cell cycle is associated with bradyzoite differentiation in Toxoplasma gondii.

We have demonstrated that bradyzoites return to the tissue-cyst stage by a developmental pathway that is indistinguishable from that initiated by sporozoites. Mature bradyzoites, like sporozoites from oocysts, were non-proliferative as they contained uniform 1N DNA contents, and replication occurred only in parasites that de-differentiated back into tachyzoites. Moreover, tachyzoites emergent from the bradyzoite-initiated pathway underwent a spontaneous growth shift prior to the onset of tissue cyst formation in a timeframe that was identical to cultures infected with sporozoites. In sporozoite-infected cultures, a novel premitotic, near-diploid subpopulation was detected during bradyzoite differentiation that co-expressed tachyzoite and bradyzoite markers. These observations suggest that activation of a G2-related cell cycle mechanism is required during bradyzoite development, and indicates that equivalent cell cycle mechanisms may govern development in the intermediate life cycle regardless of the origin of infection.

Animals↗

PubMed2002

Fundamental cellular processes do not require vertebrate-specific sequences within the TATA-binding protein.

The 180-amino acid core of the TATA-binding protein (TBPcore) is conserved from Archae bacteria to man. Vertebrate TBPs contain, in addition, a large and highly conserved N-terminal region that is not found in other phyla. We have generated a line of mice in which the tbp allele is replaced with a version, tbp(Delta N), which lacks 111 of 135 N-terminal amino acid residues. Most tbp(Delta N/Delta N) fetuses die in midgestation. To test whether a disruption of general cellular processes contributed to this fetal loss, primary fibroblast cultures were established from +/+, Delta N/+, and Delta N/Delta N fetuses. The cultures exhibited no genotype-dependent differences in proliferation or in expression of the proliferative markers dihydrofolate reductase (DHFR) mRNA (S phase-specific) and cdc25B mRNA (G(2)-specific). The mutation had no effect on transcription initiation site fidelity by either RNA polymerase II (pol II) or pol III. Moreover, the mutation did not cause differences in levels of U6 RNA, a pol III-dependent component of the splicing machinery, in mRNA splicing efficiency, in expression of housekeeping genes from either TATA-containing or TATA-less promoters, or in global gene expression. Our results indicated that general eukaryotic cell functions are unaffected by deletion of these vertebrate-specific sequences from TBP. Thus, all activities of this polypeptide domain must either be compensated for by redundant activities or be restricted to situations that are not represented by primary fibroblasts.

Animals↗

PubMed2002

Role of the E1A Rb-binding domain in repression of the NF-kappa B-dependent defense against tumor necrosis factor-alpha.

The adenoviral E1A oncogene sensitizes mammalian cells to tumor necrosis factor-alpha (TNF-alpha), in part by repressing the nuclear factor-kappa B (NF-kappa B)-dependent defense against this cytokine. Other E1A activities involve binding to either p300/cyclic AMP response element-binding protein (CBP) or retinoblastoma (Rb)-family proteins, but the roles of E1A interactions with these transcriptional regulators in sensitizing cells to TNF-alpha are unclear. E1A expression did not block upstream events in TNF-alpha-induced activation of NF-kappa B in NIH 3T3 cells, including degradation of I kappa B-alpha, nuclear translocation of NF-kappa B subunits, and their dimeric binding to kappa B sequences in the nucleus. However, E1A markedly repressed NF-kappa B-dependent transcription and sensitized cells to TNF-alpha induced apoptosis. These E1A effects were selective for kappa B-dependent transcription and for the function of the NF-kappa B p65/RelA subunit. A four amino acid E1A deletion that eliminates binding to Rb-family proteins blocked both repression of TNF-alpha-induced transcription and sensitization to apoptosis. In contrast, mutations that eliminate E1A binding to p300/CBP (coactivators of p65/RelA) did not affect either E1A activity. These data suggest that E1A-Rb-binding blocks the NF-kappa B-dependent activation response to TNF-alpha by altering the function of p65/RelA at a stage after formation of the transcription factor-enhancer complex. These observations also open questions about the general role of Rb-family proteins in modulation of NF-kappa B-dependent transcription.

3T3 Cells↗

PubMed2002

Isolation of a Toxoplasma gondii cyclin by yeast two-hybrid interactive screen.

GAL4-based yeast two-hybrid cDNA libraries from Toxoplasma gondii RH strain were constructed and screened for interactors of a putative T. gondii cdc2-related kinase, TgCRK2. A screen of 3.2 million transformants yielded a single yeast clone that harbored a protein fusion capable of specifically interacting with TgCRK2. Sequencing revealed the cDNA insert (TgCYC1) had homology to the cyclin class of proteins. The TgCYC1 cDNA fragment was used to probe a conventional T. gondii cDNA library and a 2.65 kb cDNA coding for a predicted protein of 582 amino acids was obtained. Based on comparison with a 5'-RACE product from tachyzoite mRNA, the 2.65 kb cDNA for TgCYC1 appeared to be complete. TgCYC1 had the highest similarity to Plasmodium falciparum CYC1 and displayed sequence characteristics that place it in the cyclin H class of eukaryotic cyclins. In synchronous tachyzoite populations the level of TgCYC1 mRNA was unchanged indicating it is not cell cycle regulated at the mRNA level. TgCYC1 rescues the G(1)/S cyclin cell cycle defect in S. cerevisiae strain DL1 demonstrating that this apicomplexan cyclin can function in an established heterologous model system.

Amino Acid Sequence↗