A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format.

Analysis of protein kinase C theta inhibitors for the control of HIV-1 replication in human CD4+ T cells reveals an effect on retrotranscription in addition to viral transcription.

HIV-1 infection cannot be cured due to reservoirs formed early after infection. Decreasing the massive CD4+ T cell activation that occurs at the beginning of the disease would delay reservoir seeding, providing a better prognosis for patients. CD4+ T cell activation is mediated by protein kinase C (PKC) theta (θ), which is involved in T-cell proliferation, as well as NF-κB, NF-AT, and AP-1 activation.

CGX1037 is a novel PKC isoform delta selective inhibitor in platelets.

Platelets upon activation change their shape, aggregate and secrete alpha and dense granule contents among which ADP acts as a feedback activator. Different Protein Kinase C (PKC) isoforms have specific non-redundant roles in mediating platelet responses including secretion and thrombus formation. Murine platelets lacking specific PKC isoforms have been used to evaluate the isoform specific functions.

T-cell receptor-driven lymphomagenesis in mice derived from a reprogrammed T cell.

The conversion of mature somatic cells into pluripotent stem cells, both by nuclear transfer and transduction with specific "reprogramming" genes, represents a major advance in regenerative medicine. Pluripotent stem cell lines can now be generated from an individual's own cells, facilitating the generation of immunologically acceptable stem cell-based therapeutics. Many cell types can undergo nuclear reprogramming, leading to the question of whether the identity of the reprogrammed cell of origin has a biological consequence.

Critical role of the small GTPase RhoA in the development of pulmonary edema induced by Pseudomonas aeruginosa in mice.

Background: Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia in critically ill patients. We have reported previously that P. aeruginosa exotoxins S and T mediate in vitro the increase in protein permeability across lung endothelial cell monolayers via a RhoA-dependent mechanism.

Life-cycle and growth-phase-dependent regulation of the ubiquitin genes of Trypanosoma cruzi.

Background: Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cycle that is accompanied by the stage-specific gene expression. At the molecular level, very little is known about gene regulation in trypanosomes. Complex gene organizations coupled with polycistronic transcription units make the analysis of regulated gene expression difficult in trypanosomes.

Relationship of bone morphogenetic protein expression during osteoblast differentiation to wild type p53.

We have previously shown p53 to have a specific role in osteoblast differentiation by its ability to regulate expression of certain bone specific proteins. In this study, we show mineralized matrix formation in vivo to be directly related to the presence of wild type p53 in osteoblastic osteosarcoma cells. In order to further understand the importance of p53 in differentiation, we investigated the relationship between p53 and Bone Morphogenetic Proteins (BMPs) (BMP 1, 2, 3A, 3B (GDF-10), 4, 5, 6, 7, 8A and 8B) during osteoblast differentiation.

Alternative splicing of LYT1 transcripts in Trypanosoma cruzi.

As a result of alternative trans splicing, three distinct LYT1 mRNAs are produced in Trypanosoma cruzi, two encoding the full-length LYT1 protein and the third encoding a truncated LYT1 protein lacking a possible signal sequence. Analysis of the three mRNAs in different developmental forms of the parasite revealed that the alternative processing events were regulated differently during the parasite life cycle.