Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes

The Werner and Bloom syndromes are caused by loss-of-function mutations in WRN and BLM, respectively, which encode the RecQ family DNA helicases WRN and BLM, respectively. Persons with Werner syndrome displays premature aging of the skin, vasculature, reproductive system, and bone, and those with Bloom syndrome display more limited features of aging, including premature menopause; both syndromes involve genome instability and increased cancer. The proteins participate in recombinational repair of stalled replication forks or DNA breaks, but the precise functions of the proteins that prevent rapid aging are unknown. Accumulating evidence points to telomeres as targets of WRN and BLM, but the importance in vivo of the proteins in telomere biology has not been tested. We show that Wrn and Blm mutations each accentuate pathology in later-generation mice lacking the telomerase RNA template Terc, including acceleration of phenotypes characteristic of latest-generation Terc mutants. Furthermore, pathology not observed in Terc mutants but similar to that observed in Werner syndrome and Bloom syndrome, such as bone loss, was observed. The pathology was accompanied by enhanced telomere dysfunction, including end-to-end chromosome fusions and greater loss of telomere repeat DNA compared with Terc mutants. These findings indicate that telomere dysfunction may contribute to the pathogenesis of Werner syndrome and Bloom syndrome.     

Spectroscopic properties, catalytic activities and mechanism studies of [(TpPh)Co(X)(CH3OH)m] . nCH3OH: bicarbonate dehydration in the presence of inhibitors

Two inhibitor-containing 'half-sandwich' cobalt(II) complexes [(TpPh)Co(X)(CH3OH)m] x nCH3OH ((TpPh) = hydrotris (3-phenylpyrazolyl)borate; 1: X- = N3-, m = 1, n = 2; 2: X- = NCS-, m = 0, n = 0) have been synthesized and used as the catalysts in the bicarbonate dehydration reaction. The structures of 1 and 2 were determined by X-ray diffraction analysis, which shows that N3- and NCS- coordinate to the Co(II) ions of 1 and 2, respectively, with the Co-N bond lengths of 1.992(6) A and 1.901(3) A. The coordination geometries of the Co(II) complexes in solution are five-coordinated trigonal bipyramid as revealed by the spectroscopic measurements. The dehydration kinetic measurements of HCO3- are performed by the stopped-flow techniques at pH < 7.9. The apparent dehydration rate constant k(obs) varies linearly with Co(II) complex and H+ concentrations, respectively, and the catalytic activity of 2 is lower than that of 1. The aqua Co(II) complex must be the reactive catalytic species in the catalyzed dehydration reaction and the rate-determining step is the substitution of the labile water molecule by HCO3-. The k(obs) values increase with increasing reaction temperature, and the large negative entropy of activation also indicates the associative activation mode. The inhibition ability of NCS- is stronger than that of N3-, which can be rationalized by the decreases in the Co-N(N3-/NCS-) bond lengths and effective atomic charges of the Co(II) ions based on the X-ray crystallographic data and theoretical calculations in this work.     

X-ray photoelectron spectroscopy and infrared spectroscopy study of maleimide-activated supports for immobilization of oligodeoxyribonucleotides

Surface-tethered nucleic acids are widely applied in solid-phase assays in which complementary strands must be detected against a complex mixture of other sequences. In response to such needs, numerous methods have been developed for immobilizing nucleic acids on solid supports. Often, detailed analysis of associated chemical transformations and of potential side reactions is difficult to obtain. Combined use of planar and high surface area powder supports allows characterization using surface as well as bulk diagnostic techniques. This approach is followed in the present study in which X-ray photoelectron spectroscopy (XPS), transmission infrared spectroscopy (FTIR) and reactivity titrations are used to investigate siliceous supports modified with an aminosilane precursor followed by a maleimide-bearing crosslinker for attachment of nucleic acids. The supports retain maleimide activity for approximately a day when stored under buffer, but deactivation is accelerated under basic conditions or by incomplete conversion of the precursor aminosilane monolayer. Reactions involving the olefinic bond of the imide as well as its carbonyl groups are observed and analyzed. Attachment of sulfhydryl-terminated oligodeoxyribonucleotides is highly site specific, and immobilized strands exhibit excellent hybridization activity. Quantitative use of XPS for label-free determination of DNA coverage based on calibration against reference materials is also described.     

A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains.     

Thermal chip fabrication with arrays of sensors and heaters for micro-scale impingement cooling heat transfer analysis and measurements

The design and fabrication for a thermal chip with an array of temperature sensors and heaters for study of micro-jet impingement cooling heat transfer process are presented. This thermal chip can minimize the heat loss from the system to the ambient and provide a uniform heat flux along the wall, thus local heat transfer processes along the wall can be measured and obtained. The fabrication procedure presented can reach a chip yield of 100%, and every one of the sensors and heaters on the chip is in good condition. In addition, micro-jet impingement cooling experiments are performed to obtain the micro-scale local heat transfer Nusselt number along the wall. Flow visualization for the micro-impinging jet is also made. The experimental results indicate that both the micro-scale impinging jet flow structure and the heat transfer process along the wall is significantly different from the case of large-scale jet impingement cooling process.     

Phosphorylation of p70s6 kinase is implicated in androgen-induced levator ani muscle anabolism in castrated rats

Androgens are known to increase muscle mass, strength and muscle protein synthesis. However, the molecular mechanisms by which androgens regulate skeletal muscle development remain poorly understood. The ribosomal protein kinase p70^s6k is a regulator of ribosome biogenesis and plays an important role in the regulation of growth-related protein synthesis. The phosphorylation of p70^s6k has been implicated in load-induced skeletal muscle hypertrophy. In the current study, we determined the effect of DHT on the phosphorylation of p70^s6k in the androgen-sensitive levator ani muscle of castrated rats. DHT induced a rapid increase in the phosphorylation of p70^s6k, which was detectable within 6 h after a single injection. Interestingly, DHT-induced phosphorylation of p70^s6k occurred only in androgen-sensitive muscles, but not prostate and seminal vesicle. Co-administration of flutamide, an AR antagonist, inhibited DHT-induced p70^s6k phosphorylation. While serum IGF-I levels were not changed by DHT treatment, IGF-I gene expression levels increased and the mRNA levels of IGFBP3 and IGFBP5 were suppressed in the LA muscle after DHT replacement in castrated rats. These results suggest that the phosphorylation of p70^s6k, likely via the IGF-I pathway, may play an important role in androgen-induced skeletal muscle hypertrophy.