The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long-term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell-cycle checkpoints and karyotypic stability in telomerase-expressing cells are similar to those of untransfected controls. In addition, co-expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.

Reversible covalent modification of proteins with a small ubiquitin-related modifier (SUMO) is emerging as an important system contributing to dynamic regulation of protein function. To enhance our understanding of the cell regulatory systems impacted by sumoylation, we used affinity chromatography-coupled high pressure liquid chromatography/tandem mass spectrometry for unbiased identification of candidate cellular SUMO substrate proteins. Here we describe the identification of 21 candidate sumoylated proteins from whole-cell lysates of HEK-293 cells. The nature of the proteins identified is consistent with a role for sumoylation in diverse cell regulatory systems but highlights regulation of chromatin organization and gene expression as major systems targeted by the sumoylation machinery.In addition to the well recognized role of protein phosphorylation, dynamic post-translational regulation of protein function can be achieved through a combination of a variety of other reversible covalent modifications including methylation, acetylation, ubiquitination, and sumoylation. Ubiquitination and sumoylation involve the ligation of ubiquitin family small polypeptides to one or more lysines of the target protein. Ubiquitination has long been recognized as an address code that sends proteins to the proteasome for degradation. However, apart from its role in protein degradation, ubiquitination also participates in the regulation of gene expression, signal transduction, and intracellular transport. The consequences of sumoylation on target proteins are relatively obscure; although based on the growing list of identified sumoylated proteins, sumoylation has been implicated in a diverse array of cell regulatory functions including the regulation of chromatin structure, subcellular compartmentalization, transcription factor activity, DNA binding, and protein complex assembly (1-3).There are three known small ubiquitin-like modifier (SUMO) 1 family members in mammalian cells: SUMO1, -2, and -3. The biochemical pathway mediating protein sumoylation has been characterized and is analogous to that defined for ubiquitination. Namely, the combined action of three proteins (an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase) mediates isopeptide bond formation between SUMO and the target lysine on the substrate protein. Importantly, sumoylation is reversible, and a number of SUMO proteases that can cleave SUMO from specific target proteins have been identified (1-3).The bulk of present knowledge of the role of sumoylation in the regulation of cell behavior has been inferred from the nature of identified SUMO substrate proteins (3). The list of known SUMO substrates has been compiled by a combination of serendipitous observations and direct candidate analysis. It is not at all clear how well this list reflects the contribution of dynamic sumoylation to cell regulatory events. Therefore, we wished to assess the feasibility of a proteomics-based approach for an unbiased sampling of cellular SUMO target proteins. Here, ...

No abstract

Genetic dissection of nucleoside transport in Leishmiania dotnov alni indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 ,uM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 puM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.The parasitic protozoa are the causative agents for a plethora of infectious diseases. Most of the major metabolic pathways in parasites are thought to be similar to those found in mammalian systems with one major exception. All the parasitic protozoa examined to date are incapable of de novo synthesis of the purine ring and are therefore auxotrophic for purines (14). Cultured forms of parasites are dependent on an exogenous source of purines for survival and growth, whereas intracellular parasites scavenge purines from their hosts. To meet their purine requirement, the parasites have evolved a series of unique purine salvage enzymes and cell surface functions for which no mammalian counterpart exists. Because nucleoside or nucleobase analogs are selectively metabolized by this unique purine salvage system, they are growth inhibitory and cytotoxic to parasitic protozoa but not to mammalian cells. For instance, allopurinol (4-hydroxypyrazolo[3,4,-d]pyrimnidine) (1, 15), allopurinol riboside (1, 19), thiopurinol (4-thiopyrazolo[3,4-d] pyrimidine) (15), thiopurinol riboside (15), and formycin B (7-hydroxy-3-,-D-ribofuranosyl-pyrazolo[4,3-d]pyrimidine)

Ral GTPases have been implicated as mediators of Ras-induced signal transduction from observations that

Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been demonstrated to be an effective agent against a variety of parasitic protozoa but not against Leishmania spp. In this report, we show that Leishmania donovani promastigotes in continuous culture are sensitive to the growth inhibitory and cytotoxic effects of DFMO. Incubation of the promastigotes with DFMO obliterates intracellular putrescine pools and depletes spermidine concentrations, which correlates with the onset of growth inhibition. The effects of DFMO on the growth and the intracellular polyamine pools can be reversed completely by the addition of 10 microM putrescine to the culture medium. These results suggest that the treatment of leishmaniasis may be amenable to chemotherapeutic manipulation by DFMO.

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach selecting for defective vocalization in mice, we identified Kdm5a as a candidate ASD gene. To validate our discovery, we generated a Kdm5a knockout mouse model (Kdm5a-/-) and confirmed that inactivating Kdm5a disrupts vocalization. In addition, Kdm5a-/- mice displayed repetitive behaviors, sociability deficits, cognitive dysfunction, and abnormal dendritic morphogenesis. Loss of KDM5A also resulted in dysregulation of the hippocampal transcriptome. To determine if KDM5A mutations cause ASD in humans, we screened whole exome sequencing and microarray data from a clinical cohort. We identified pathogenic KDM5A variants in nine patients with ASD and lack of speech. Our findings illustrate the power and efficacy of forward genetics in identifying ASD genes and highlight the importance of KDM5A in normal brain development and function.