Serum Response Factor Regulates Immediate Early Host Gene Expression in Toxoplasma gondii-Infected Host Cells

Toxoplasma gondii is a wide spread pathogen that can cause severe and even fatal disease in fetuses and immune-compromised hosts. As an obligate intracellular parasite, Toxoplasma must alter the environment of its host cell in order to establish its replicative niche. This is accomplished, in part, by secretion of factors into the host cell that act to modulate processes such as transcription. Previous studies demonstrated that genes encoding transcription factors such as c-jun, junB, EGR1, and EGR2 were amongst the host genes that were the most rapidly upregulated following infection. In cells stimulated with growth factors, these genes are regulated by a transcription factor named Serum Response Factor. Serum Response Factor is a ubiquitously expressed DNA binding protein that regulates growth and actin cytoskeleton genes via MAP kinase or actin cytoskeletal signaling, respectively. Here, we report that Toxoplasma infection leads to the rapid activation of Serum Response Factor. Serum Response Factor activation is a Toxoplasma-specific event since the transcription factor is not activated by the closely related protozoan parasite, Neospora caninum. We further demonstrate that Serum Response Factor activation requires a parasite-derived secreted factor that signals via host MAP kinases but independently of the host actin cytoskeleton. Together, these data define Serum Response Factor as a host cell transcription factor that regulates immediate early gene expression in Toxoplasma-infected cells

Gene discovery in the Apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genome Res

Large-scale EST sequencing projects for several important parasites within the phylum Apicomplexa were undertaken for the purpose of gene discovery. Included were several parasites of medical importance (Plasmodium falciparum, Toxoplasma gondii) and others of veterinary importance (Eimeria tenella, Sarcocystis neurona, and Neospora caninum). A total of 55,192 ESTs, deposited into dbEST/GenBank, were included in the analyses. The resulting sequences have been clustered into nonredundant gene assemblies and deposited into a relational database that supports a variety of sequence and text searches. This database has been used to compare the gene assemblies using BLAST similarity comparisons to the public protein databases to identify putative genes. Of these new entries, ∼15%-20% represent putative homologs with a conservative cutoff of p < 10 −9 , thus identifying many conserved genes that are likely to share common functions with other well-studied organisms. Gene assemblies were also used to identify strain polymorphisms, examine stage-specific expression, and identify gene families. An interesting class of genes that are confined to members of this phylum and not shared by plants, animals, or fungi, was identified. These genes likely mediate the novel biological features of members of the Apicomplexa and hence offer great potential for biological investigation and as possible therapeutic targets

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

Gene Discovery in the Apicomplexa as Revealed by EST Sequencing and Assembly of a Comparative Gene Database

Large-scale EST sequencing projects for several important parasites within the phylum Apicomplexa were undertaken for the purpose of gene discovery. Included were several parasites of medical importance (Plasmodium falciparum, Toxoplasma gondii) and others of veterinary importance (Eimeria tenella, Sarcocystis neurona, and Neospora caninum). A total of 55,192 ESTs, deposited into dbEST/GenBank, were included in the analyses. The resulting sequences have been clustered into nonredundant gene assemblies and deposited into a relational database that supports a variety of sequence and text searches. This database has been used to compare the gene assemblies using BLAST similarity comparisons to the public protein databases to identify putative genes. Of these new entries, ∼15%–20% represent putative homologs with a conservative cutoff of p < 10(−9), thus identifying many conserved genes that are likely to share common functions with other well-studied organisms. Gene assemblies were also used to identify strain polymorphisms, examine stage-specific expression, and identify gene families. An interesting class of genes that are confined to members of this phylum and not shared by plants, animals, or fungi, was identified. These genes likely mediate the novel biological features of members of the Apicomplexa and hence offer great potential for biological investigation and as possible therapeutic targets. [The sequence data from this study have been submitted to dbEST division of GenBank under accession nos.: Toxoplasma gondii: –, –, –, –, – , –, –, –, –. Plasmodium falciparum: –, –, –, –. Sarcocystis neurona: , , , , , , , , , , , , , –, –, –, –, –. Eimeria tenella: –, –, –, –, –, –, –, –, – , –, –, –, –, –, –, –, –, –, –, –. Neospora caninum: –, –, , – , –, –.

Continuous Lenalidomide Treatment for Newly Diagnosed Multiple Myeloma

Background Lenalidomide has tumoricidal and immunomodulatory activity against multiple myeloma. This double-blind, multicenter, randomized study compared melphalan-prednisone-lenalidomide induction followed by lenalidomide maintenance (MPR-R) with melphalan-prednisone-lenalidomide (MPR) or melphalan-prednisone (MP) followed by placebo in patients 65 years of age or older with newly diagnosed multiple myeloma. Methods We randomly assigned patients who were ineligible for transplantation to receive MPR-R (nine 4-week cycles of MPR followed by lenalidomide maintenance therapy until a relapse or disease progression occurred [152 patients]) or to receive MPR (153 patients) or MP (154 patients) without maintenance therapy. The primary end point was progression-free survival. Results The median follow-up period was 30 months. The median progression-free survival was significantly longer with MPR-R (31 months) than with MPR (14 months; hazard ratio, 0.49; P&lt;0.001) or MP (13 months; hazard ratio, 0.40; P&lt;0.001). Response rates were superior with MPR-R and MPR (77% and 68%, respectively, vs. 50% with MP; P&lt;0.001 and P=0.002, respectively, for the comparison with MP). The progression-free survival benefit associated with MPR-R was noted in patients 65 to 75 years of age but not in those older than 75 years of age (P=0.001 for treatment-by-age interaction). After induction therapy, a landmark analysis showed a 66% reduction in the rate of progression with MPR-R (hazard ratio for the comparison with MPR, 0.34; P&lt;0.001) that was age-independent. During induction therapy, the most frequent adverse events were hematologic; grade 4 neutropenia was reported in 35%, 32%, and 8% of the patients in the MPR-R, MPR, and MP groups, respectively. The 3-year rate of second primary tumors was 7% with MPR-R, 7% with MPR, and 3% with MP. Conclusions MPR-R significantly prolonged progression-free survival in patients with newly diagnosed multiple myeloma who were ineligible for transplantation, with the greatest benefit observed in patients 65 to 75 years of age