Telomerase-dependent and -independent chromosome healing in mouse embryonic stem cells

Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have previously demonstrated that double-strand breaks near telomeres in mammalian cells result in either the addition of a new telomere at the site of the break, termed chromosome healing, or sister chromatid fusion that initiates chromosome instability. In the present study, we have investigated the role of telomerase in chromosome healing and the importance of chromosome healing in preventing chromosome instability. In embryonic stem cell lines that are wild type for the catalytic subunit of telomerase (TERT), chromosome healing at I-SceI-induced double-strand breaks near telomeres accounted for 22 of 35 rearrangements, with the new telomeres added directly at the site of the break in all but one instance. In contrast, in two TERT-knockout embryonic stem cell lines, chromosome healing accounted for only 1 of 62 rearrangements, with a 23 bp insertion at the site of the sole chromosome-healing event. However, in a third TERT-knockout embryonic stem cell line, 10PTKO-A, chromosome healing was a common event that accounted for 20 of 34 rearrangements. Although this chromosome healing also occurred at the I-SceI site, differences in the microhomology at the site of telomere addition demonstrated that the mechanism was distinct from that in wild-type embryonic stem cell lines. In addition, the newly added telomeres in 10PTKO-A shortened with time in culture, eventually resulting in either telomere elongation through a telomerase-independent mechanism or loss of the subtelomeric plasmid sequences entirely. The combined results demonstrate that chromosome healing can occur through both telomerase-dependent and -independent mechanisms, and that although both mechanisms can prevent degradation and sister chromatid fusion, neither mechanism is efficient enough to prevent sister chromatid fusion from occurring in many cells experiencing double-strand breaks near telomeres.     

Phylogeny of the New World true frogs (Rana)

Phylogenetic relationships among the species of true frogs (Rana) from North, South, and Central America were investigated based on the sequences of approximately 2 kb from the mitochondrial genome, sampled from most of the described species, as well as eight undescribed species. This analysis, combined with previous studies of the phylogeny of New World Rana, served as the basis for a revised classification of the group. The American species of Rana are not monophyletic; the western North American Amerana is more closely related to the R. temporaria group of Eurasia (together, these frogs form the group Laurasiarana). The remaining species from the Americas form the monophyletic group Novirana, which includes: R. sylvatica; Aquarana (the R. catesbeiana group); Ranula (the R. palmipes group, including the mostly upland Levirana species and the mostly lowland Lithobates species); Torrentirana (the R. tarahumarae group, or Zweifelia, plus R. sierramadrensis), Stertirana (the R. montezumae group, or Lacusirana, plus R. pipiens), Nenirana (the R. areolata group), and Scurrilirana (most of the southern and tropical leopard frogs). The mitochondrial sequences supported many of the previous hypotheses of relationships of New World Rana, although there were some differences involving the placement of the species R. pipiens, R. sierramadrensis, and R. sylvatica. Parametric bootstrap analyses indicated significant support for the relationships inferred from the mtDNA sequences, and rejected the previous hypotheses of relationships for these three species.     

Whole-genome variance components linkage analysis using single-nucleotide polymorphisms versus microsatellites on quantitative traits of derived phenotypes from factor analysis of electroencephalogram waves

Alcohol dependence is a serious public health problem. We studied data from families participating in the Collaborative Study on the Genetics of Alcoholism (COGA) and made available to participants in the Genetic Analysis Workshop 14 (GAW14) in order to search for genes predisposing to alcohol dependence. Using factor analysis, we identified four factors (F1, F2, F3, F4) related to the electroencephalogram traits. We conducted variance components linkage analysis with each of the factors. Our results using the Affymetrix single-nucleotide polymorphism dataset showed significant evidence for a novel linkage of F3 (factor comprised of the three midline channel EEG measures from the target case of the Visual Oddball experiment ttdt2, 3, 4) to chromosome 18 (LOD = 3.45). This finding was confirmed by analyses of the microsatellite data (LOD = 2.73) and Illumina SNP data (LOD = 3.30). We also demonstrated that, in a sample like the COGA data, a dense single-nucleotide polymorphism map provides better linkage signals than low-resolution microsatellite map with quantitative traits.     

Angiotensin-induced defects in renal oxygenation: role of oxidative stress

We tested the hypothesis that superoxide anion (O(2)(-).) generated in the kidney by prolonged angiotensin II (ANG II) reduces renal cortical Po(2) and the use of O(2) for tubular sodium transport (T(Na):Q(O(2))). Groups (n = 8-11) of rats received angiotensin II (ANG II, 200 ng.kg(-1).min(-1) sc) or vehicle for 2 wk with concurrent infusions of a permeant nitroxide SOD mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol, 200 nmol.kg(-1).min(-1)) or vehicle. Rats were studied under anesthesia with measurements of renal oxygen usage and Po(2) in the cortex and tubules with a glass electrode. Compared with vehicle, ANG II increased mean arterial pressure (107 +/- 4 vs. 146 +/- 6 mmHg; P < 0.001), renal vascular resistance (42 +/- 3 vs. 65 +/- 7 mmHg.ml(-1).min(-1).100 g(-1); P < 0.001), renal cortical NADPH oxidase activity (2.3 +/- 0.2 vs. 3.6 +/- 0.4 nmol O(2)(-)..min(-1).mg(-1) protein; P < 0.05), mRNA and protein expression for p22(phox) (2.1- and 1.8-fold respectively; P < 0.05) and reduced the mRNA for extracellular (EC)-SOD (-1.8 fold; P < 0.05). ANG II reduced the Po(2) in the proximal tubule (39 +/- 1 vs. 34 +/- 2 mmHg; P < 0.05) and throughout the cortex and reduced the T(Na):Q(O(2)) (17 +/- 1 vs. 9 +/- 2 mumol/mumol; P < 0.001). Tempol blunted or prevented all these effects of ANG II. The effects of prolonged ANG II to cause hypertension, renal vasoconstriction, renal cortical hypoxia, and reduced efficiency of O(2) usage for Na(+) transport, activation of NADPH oxidase, increased expression of p22(phox), and reduced expression of EC-SOD can be ascribed to O(2)(-). generation because they are prevented by an SOD mimetic.     

Rapamycin analogs with reduced systemic exposure

The synthesis and biological activities of rapamycin (I) analogs modified at the C-40 position are reported. Emphasis placed on compounds that potentially have an improved safety profile on account of their shorter in vivo half-life when compared with rapamycin.     

A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.     

Cutting edge: Expression of functional CD137 receptor by dendritic cells

Interaction between dendritic cells (DCs) and T cells is a prerequisite for the initiation of a T cell response. The molecular nature of this interaction remains to be fully characterized. We report in this work that freshly isolated mouse splenic DCs and bone marrow-derived DCs express CD137 on the cell surface and in soluble form. Triggering CD137 increased the secretion of IL-6 and IL-12 from DCs. More importantly, infusion of an agonistic mAb to CD137 into naive mice enhanced the ability of DCs to stimulate T cell proliferation in response to both alloantigens and a nominal Ag in vitro. This enhancement of DC function is not mediated through activation of T cells, because the effect was also observed in RAG-1 knockout mice that lack T cells. Our findings implicate CD137 as an important receptor involved in the modulation of DC function.