Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.     

Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response

The stabilization and processing of stalled replication forks is required to maintain genome integrity in all organisms. In an effort to identify novel proteins that might be involved in stabilizing stalled replication forks, Saccharomyces cerevisiae mutant wss1Δ was isolated from a high-throughput screening of ~5000 deletion strains for genes involved in the response to continuous, low-intensity UV irradiation. Disruption of WSS1 resulted in synergistic increases in UV sensitivity with null mutants of genes involved in recombination (RAD52) and cell cycle control (RAD9 and RAD24). WSS1 was also found to interact genetically with SGS1, TOP3, SRS2 and CTF4, which are involved in recombination, repair of replication forks and the establishment of sister chromatid cohesion. A yeast two-hybrid screen identified a potential physical interaction between Wss1 and both Psy2 and Tof1. Genetic interactions were also detected between PSY2 and TOF1, as well as between each gene and RAD52 and SRS2, and between WSS1 and TOF1. Tof1 is known to be involved in stabilizing stalled replication forks and our data suggest that Wss1 and Psy2 similarly function to stabilize or process stalled or collapsed replication forks.     

Calpains mediate integrin attachment complex maintenance of adult muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line- or M-line-specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans.     

Improving clinical outcomes using adoptively transferred immune cells from umbilical cord blood

Because of the necessary immunodepletion prior to cord blood transplantation as well as the immaturity of cord blood immune cells, recipients experience a high incidence of viral infection in addition to complications observed after hematopoietic stem cell transplantation, such as relapse and graft-versus-host disease. We describe current immunotherapeutic approaches to treating these complications, including the generation of antigen-specific T cells from cord blood, redirecting cord blood T cells using chimeric antigen receptors, and generating cord blood-derived natural killer cells and regulatory T cells.     

Isolation and identification of eight microsatellite loci in the Cherokee darter (Etheostoma scotti) and their variability in other members of the genera Etheostoma, Ammocrypta, and Percina

The Cherokee darter Etheostoma scotti is a federally threatened fish endemic to the Etowah River system of northwest Georgia. In order to analyse the population structure and genetic diversity of this fish, eight tetranucleotide microsatellite genetic markers were developed. The marker set was applied to 13 additional darter species to test cross-species amplification and polymorphism. Successful amplification was obtained for all eight loci in each of the 13 other species of darters, with between seven and eight polymorphic loci per species.