Functional link between BLM defective in Bloom's syndrome and the ataxia-telangiectasia-mutated protein,ATM

Chromosome aberrations, genomic instability, and cancer predisposition are hallmarks of a number of syndromes in which the defective genes recognize and/or repair DNA damage or are involved in some aspect of DNA processing. We report here direct interaction between BLM, mutated in Bloom's Syndrome (BS), and ATM, mutated is ataxia-telangiectasia, and we have mapped the sites of interaction. Full-length BLM cDNA corrected sister chromatid exchange (SCE) and radiosensitivity in BS cells. Mitotic phosphorylation of BLM was partially dependent on ATM, and phosphorylation sites on BLM were identified. A phosphospecific antibody against one of these sites (Thr-99) revealed radiation-induced phosphorylation, which was defective in ataxia-telangiectasia cells. Stable cell lines expressing phosphorylation site mutants failed to correct radiosensitivity in BS cells but corrected SCE. These mutants also sensitized normal control cells to radiation and increased radiation-induced chromosome aberrations but did not cause SCE numbers to increase. These data suggest that ATM and BLM function together in recognizing abnormal DNA structures by direct interaction and that these phosphorylation sites in BLM are important for radiosensitivity status but not for SCE frequency.     

Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.     

Distribution and abundance of euphausiids and pelagic amphipods in Kongsfjorden,Isfjorden and Rijpfjorden (Svalbard) and changes in their relative importance as key prey in a warming marine ecosystem

Euphausiid (krill) and amphipod dynamics were studied during 2006–2011 by use of plankton nets in Kongsfjorden (79°N) and adjacent waters, also including limited sampling in Isfjorden (78°N) and Rijpfjorden (80°N). The objectives of the study were to assess how variations in physical characteristics across fjord systems affect the distribution and abundance of euphausiids and amphipods and the potential for these macrozooplankton species to reproduce in these waters. The abundances of euphausiids and amphipods were higher in Kongsfjorden than in Rijpfjorden and Isfjorden, and the highest abundances were observed at the innermost stations of Kongsfjorden, where Thysanoessa inermis and Themisto libellula dominated. The Atlantic species Thysanoessa longicaudata, Meganyctiphanes norvegica and Themisto abyssorum dominated at the outside Kongsfjorden. Inter-annual and seasonal variability in abundances of euphausiids and amphipods were evident. The presence of ripe euphausiids outside Kongsfjorden indicates that they may reproduce in these areas. Mature individuals of T. abyssorum were recorded mainly outside Kongsfjorden, whereas no mature or ripe T. libellula were present in both the inner and outer parts of this fjord. If the warming trend persists, as seen during the last decade, this would favour the Atlantic/boreal euphausiid species, while Arctic species, such as the amphipod T. libellula, may decline. Euphausiids and amphipods are major food of capelin (Mallotus villosus) and polar cod (Boreogadus saida), respectively, in this region, and changes in prey abundance will likely have an impact on the feeding dynamics of these important fish species.     

Anthropogenic signature in the incidence and distribution of an emerging pathogen of poplars

The introduction and establishment of non-native plant pathogens into new areas can result in severe outbreaks. Septoria leaf spot and canker caused by Sphaerulina musiva is one of the most damaging poplar diseases in northeastern and north-central North America. Stem and branch cankers can be devastating on susceptible trees, leading to tree death and reduced biomass in commercial plantations. In the Pacific Northwest region of North America, the first report of the disease was made in 2006 in the Fraser Valley of British Columbia (BC), Canada. To investigate the incidence and distribution of S. musiva from its point of introduction into BC, five plantations of Populus trichocarpa (black cottonwood), 500 P. trichocarpa trees from natural populations, and 23 plantations of hybrid poplars were surveyed by using real-time PCR assays targeting S. musiva and its native sister species, S. populicola. Our survey suggests a strong anthropogenic signature to the emergence of the non-native S. musiva. Detection frequency of S. musiva was high in hybrid poplar plantations (116 trees infected, 54.2 % of the sampled trees), while detection of the native S. populicola was limited to 13.1 % (22 trees infected). By contrast, in natural stands of P. trichocarpa, less than 2 % of the trees were positive for S. musiva (7 trees) while ~75 % were positive for S. populicola (433 trees). All the S. musiva detections in natural stands of the native P. trichocarpa were from trees located in the vicinity (<2.5 km) of hybrid poplar plantations. Identification of the genotypes found in the hybrid poplar plantations revealed that they are in majority F1 progeny from P. trichocarpa × P. deltoides (T × D) (82 %) and P. nigra × P. maximowiczii (N × M) (7.8 %) crosses, which are generally susceptible (intermediate level of susceptibility between the two parental species) to the canker disease. Our results suggest that the emergence of S. musiva in BC is related to the planting of susceptible hybrid poplars. Even if the disease has not yet established itself in natural poplar populations outside of the Fraser Valley, infected plantations could act as a reservoir that could promote its spread into nearby native P. trichocarpa populations.     

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57%+/-4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.     

HMGB1 is a cofactor in mammalian base excision repair

Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.     

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.     

A naturally occurring calcineurin variant inhibits FoxO activity and enhances skeletal muscle regeneration

The calcium-activated phosphatase calcineurin (Cn) transduces physiological signals through intracellular pathways to influence the expression of specific genes. Here, we characterize a naturally occurring splicing variant of the CnAβ catalytic subunit (CnAβ1) in which the autoinhibitory domain that controls enzyme activation is replaced with a unique C-terminal region. The CnAβ1 enzyme is constitutively active and dephosphorylates its NFAT target in a cyclosporine-resistant manner. CnAβ1 is highly expressed in proliferating myoblasts and regenerating skeletal muscle fibers. In myoblasts, CnAβ1 knockdown activates FoxO-regulated genes, reduces proliferation, and induces myoblast differentiation. Conversely, CnAβ1 overexpression inhibits FoxO and prevents myotube atrophy. Supplemental CnAβ1 transgene expression in skeletal muscle leads to enhanced regeneration, reduced scar formation, and accelerated resolution of inflammation. This unique mode of action distinguishes the CnAβ1 isoform as a candidate for interventional strategies in muscle wasting treatment.     

Effect of dietary copper on selenium toxicity in Fischer 344 rats

The purpose of this study was to investigate the ameliorating effects of dietary copper supplementation on selenium toxicity. Nine groups (n = 6) of weanling Fischer 344 female rats were randomly assigned to treatment groups and fed diets containing nontoxic levels of copper as CuCl2 and/or selenium as selenite or selenocystamine. Weight gain, liver and spleen weights, plasma lipid peroxidation, and liver selenium and copper content were analyzed after the 6-wk treatment period. Concentrations of up to 10 times the daily lethal dose of dietary selenium were well tolerated in rats supplemented with dietary copper. As the dietary level of selenium was increased, the ratio of selenium to copper measured in the liver decreased. In the groups of rats in which dietary copper supplementation was absent and dietary selenium was supplemented, copper stores in the liver remained unchanged from control values. Copper's protective effects from dietary selenium toxicity may come from the formation of a copper-selenide complex that renders both selenium and copper metabolically unavailable and nontoxic.