Variant repeats are interspersed throughout the telomeres and recruit nuclear receptors in ALT cells

Telomeres in cells that use the recombination-mediated alternative lengthening of telomeres (ALT) pathway elicit a DNA damage response that is partly independent of telomere length. We therefore investigated whether ALT telomeres contain structural abnormalities that contribute to ALT activity. Here we used next generation sequencing to analyze the DNA content of ALT telomeres. We discovered that variant repeats were interspersed throughout the telomeres of ALT cells. We found that the C-type (TCAGGG) variant repeat predominated and created a high-affinity binding site for the nuclear receptors COUP-TF2 and TR4. Nuclear receptors were directly recruited to telomeres and ALT-associated characteristics were induced after incorporation of the C-type variant repeat by a mutant telomerase. We propose that the presence of variant repeats throughout ALT telomeres results from recombination-mediated telomere replication and spreading of variant repeats from the proximal regions of the telomeres and that the consequent binding of nuclear receptors alters the architecture of telomeres to facilitate further recombination.     

The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.     

Moderate chemical modifications of WAY-100635 improve the selectivity for 5-HT1A versus D4 receptors

The selectivity for 5-HT(1A) versus D(4) receptors is significantly increased when the basic side chain of WAY-100635 is replaced by a 4-phenylpiperazine (3e) or a 4-phenyl-1,2,3,6-tetrahydropyridine moiety (3i). The 4-phenyl-1,2,3,6-tetrahydropyridine compounds (3i-l) have a higher affinity for 5-HT(1A) receptors than do the corresponding unsubstituted phenylpiperazine analogues (3e-h). Compounds 3e and 3i appear to be selective for 5-HT(1A) receptors over other relevant receptors and still behave as neutral antagonists.     

Comparison of Bacteria and Archaea communities in municipal solid waste, individual refuse components, and leachate

Refuse decomposition in landfills is a microbially mediated process that occurs primarily under anaerobic conditions. Because of limited moisture conditions, hydraulic transport as a means of cellular translocation within the landfill appears limited, especially during the initial stages of decomposition. Thus, microbial communities within the incoming refuse serve as a primary source of facultative and obligate anaerobic microorganisms that initiate refuse decomposition. Fresh residential refuse was collected five times over 26 months, and microbial communities in these samples were compared with those in individual refuse components and decomposed refuse. Bacterial and archaeal community structures were determined using T-RFLP. The Bacterial microbial community richness was correlated (r(2) = 0.91) with seasonal differences in ambient air temperature. Analysis of the results shows that fresh refuse is most likely not the source of methanogens in landfills. Microbial communities in the solid and leachate phases were different, indicating that both matrices must be considered when characterizing microbial diversity within a landfill.     

Silencing the activity and proliferative properties of the human EagI Potassium Channel by RNA Interference

EagI potassium channels are natively expressed in the mammalian brain as well as in many cancer cell lines and tumor tissues. The role of EagI in malignant transformation has been suggested by several experiments, but the lack of specific EagI inhibitors has made it difficult to examine the influence of the channel on oncogenesis and its potential as a therapeutic target. We have used short interfering RNA to test the effects of EagI reduction on the behavior of tumor cells in vitro. By generating and optimizing an EagI-specific short interfering RNA system, we were able to study the effects of EagI depletion on several cancer cell lines that endogenously express this protein. We show here that our short interfering RNA sequences act specifically on EagI, reproducibly induce a significant decrease in the proliferation of tumor cell lines, and do not trigger any observable nonspecific responses.     

Hyaluronan constitutively regulates activation of multiple receptor tyrosine kinases in epithelial and carcinoma cells

Hyaluronan (HA) is enriched in the pericellular matrices of many malignant human tumors, and manipulations of HA interactions have strong effects on tumor progression in animal models. Increased HA production stimulates ERBB2 activation, leading to increased cell survival activities and several malignant cell properties. On the other hand, inhibition of constitutive HA-tumor cell interactions in malignant cells inhibits these properties. We have now investigated the role of HA in activation of several additional receptor tyrosine kinases (RTKs), i.e. IGF1R-beta, PDGFR-beta, EGFR and c-MET, in colon, prostate, and breast carcinoma cells. In each case we show that antagonists of endogenous HA interactions inhibit their tyrosine phosphorylation, i.e. activation. On the other hand, we show that these RTKs are activated in phenotypically normal or relatively benign tumor cells by experimentally increasing HA production. We also investigated the role of HA in constitutive versus ligand-induced activation of RTKs. In HCA7 colon and C4-2 prostate carcinoma cells, ERBB2 is constitutively activated in a ligand-independent manner, whereas IGF1R-beta and PDGFR-beta require ligand interaction for activation. We show that both constitutive activation of ERBB2 and ligand-mediated activation of IGF1R-beta and PDGFR-beta are reversed by co-treatment of the cells with a HA antagonist. We conclude that HA serves a general function in RTK activation.     

Regulated expression of FLRT genes implies a functional role in the regulation of FGF signalling during mouse development

Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.     

Normal and abnormal fetal growth

The fetal origins of adult disease hypothesis suggests that poor intrauterine growth is associated with an increased risk of cardiovascular disease. The hypothesis goes on to implicate different growth 'phenotypes', particularly disproportionate growth, in the determination of the type of cardiovascular disease that develops. Analysis of the antenatal growth of a low-risk pregnancy population does not identify such growth phenotypes within the general population. Rather, intrauterine growth is characterized by poor predictability of subsequent size, suggesting that centile crossing is a common feature of intrauterine growth. Furthermore, there is a sexually dimorphic pattern to this growth that needs to be considered in further work to test the fetal origins hypothesis.