Human chromosome 5 complements the DNA double-strand break-repair deficiency and gamma-ray sensitivity of the XR-1 hamster variant.

XR-1 is a Chinese hamster ovary (CHO) cell mutant which is unusually sensitive to killing by gamma rays in the G1 portion of the cell cycle but has nearly normal resistance to gamma-ray damage in late S phase. The cell-cycle sensitivity correlates with the mutant's inability to repair DNA double-strand breaks (DSBs) produced by ionizing radiation and restriction enzymes. We have previously shown in somatic cell hybrids of XR-1 cells and human fibroblasts that the XR-1 mutation is a recessive mutation. In this study, using somatic cell hybrids formed between XR-1 and human fibroblasts, we map the human complementing gene to chromosome 5 by chromosome-segregation analysis. This gene biochemically restores the hamster defect to wild-type levels of gamma-ray and bleomycin resistance as well as restoring its proficiency to repair DNA DSBs, suggesting that a single gene is responsible for the XR-1 phenotype. We have tentatively assigned the name XRCC4 (X-ray-complementing Chinese hamster gene 4) to this human gene until its biochemical function in repair is discovered.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Cell-cycle-dependent repair of potentially lethal damage in the XR-1 gamma-ray-sensitive Chinese hamster ovary cell

Repair of potentially lethal damage (PLD) was investigated in a gamma-ray-sensitive Chinese hamster cell mutant, XR-1, and its parent by comparing survival of plateau-phase cells plated immediately after irradiation with cells plated after a delay. Previous work indicated that XR-1 cells are deficient in repair of double-strand DNA breaks and are gamma-ray sensitive in G1 but have near normal sensitivity and repair capacity in late S phase. At irradiation doses from 0 to 1.0 Gy (100 to 10% survival), the delayed- and immediate-plating survival curves of XR-1 cells were identical; however, at doses greater than 1.0 Gy a significant increase in survival was observed when plating was delayed (PLD repair), approaching a 20-fold increase at 8 Gy. Elimination of S-phase cells by [3H]thymidine suicide dramatically increased gamma-ray sensitivity of plateau-phase XR-1 mutant cells and reduced by 600-fold the number of cells capable of PLD repair after a 6-Gy dose. In contrast, elimination of S-phase cells in plateau-phase parental cells did not alter PLD repair. These results suggest that the majority of PLD repair observed in plateau-phase XR-1 cells occurs in S-phase cells while G1 cells perform little PLD repair. In contrast, G1 cells account for the majority of PLD repair in plateau-phase parental cells. Thus, in the XR-1 mutant, a cell's ability to repair PLD seems to depend upon the stage of the cell cycle at which the irradiation is delivered. A possible explanation for these findings is discussed.     

HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.     

CDC2/SPDY transiently associates with endoplasmic reticulum exit sites during oocyte maturation

Background  

Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.     

Mouse models to unravel the role of inhaled pollutants on allergic sensitization and airway inflammation

Background

Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.

Methods

Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca²⁺]_i) were conducted.

Results

Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca²⁺]_i revealed changes in membrane current in response to ACh and in [Ca²⁺]_i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca²⁺]_i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium.

Conclusions

Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca²⁺-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.     

Local erythropoietin and endothelial progenitor cells improve regional cardiac function in acute myocardial infarction

Background

Expanded endothelial progenitor cells (eEPC) improve global left ventricular function in experimental myocardial infarction (MI). Erythropoietin beta (EPO) applied together with eEPC may improve regional myocardial function even further by anti-apoptotic and cardioprotective effects. Aim of this study was to evaluate intramyocardial application of eEPCs and EPO as compared to eEPCs or EPO alone in experimental MI.

Methods and Results

In vitro experiments revealed that EPO dosed-dependently decreased eEPC and leukocyte apoptosis. Moreover, in the presence of EPO mRNA expression in eEPC of proangiogenic and proinflammatory mediators measured by TaqMan PCR was enhanced. Experimental MI was induced by ligation and reperfusion of the left anterior descending coronary artery of nude rats (n = 8-9). After myocardial transplantation of eEPC and EPO CD68+ leukocyte count and vessel density were enhanced in the border zone of the infarct area. Moreover, apoptosis of transplanted CD31 + TUNEL + eEPC was decreased as compared to transplantation of eEPCs alone. Regional wall motion of the left ventricle was measured using Magnetic Resonance Imaging. After injection of eEPC in the presence of EPO regional wall motion significantly improved as compared to injection of eEPCs or EPO alone.

Conclusion

Intramyocardial transplantation of eEPC in the presence of EPO during experimental MI improves regional wall motion. This was associated with an increased local inflammation, vasculogenesis and survival of the transplanted cells. Local application of EPO in addition to cell therapy may prove beneficial in myocardial remodeling.