Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1⁺ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.     

Effects of density, climate, and supplementary forage on body mass and pregnancy rates of female red deer in Spain

The influence of short- and long-term (cohort) effects of climate and density on the life-histories of ungulates in temperate regions may vary with latitude, habitat, and management practices, but the life-histories of ungulates in the Mediterranean region are less well known. This study examined the short- and long-term effects of rainfall and absolute density on hinds in two of the southernmost populations of red deer (Cervus elaphus hispanicus) in Europe. One population received supplementary forage. Unlike more northerly latitudes, where red deer hinds lose body mass in winter as a result of adverse weather, in the Spanish populations, hinds did not lose body mass. Hinds in the population that received supplementary forage were heavier and more likely to become pregnant than were the hinds in the unsupplemented population. The likelihood of pregnancy occurring was strongly influenced by hind body mass; the proportion of yearlings that became pregnant was consequently lower in the unsupplemented population than in the population that received supplementary forage. Cohort effects on hind body mass (negative for density and positive for rainfall at birth) and on the probability of pregnancy (negative for density at birth) were apparent only in the unsupplemented population, which implies that supplemental feeding may partially compensate for negative density-dependent factors during early growth, and that supplemented deer hinds may experience reduced selection pressures. These results reflect the particular seasonal variation in the abundance and quality of food in Mediterranean habitats. The delayed effects of climate and density at birth on adult hind body mass and the prevalence of pregnancy probably affects population dynamics and constitutes a mechanism by which cohort effects affect the population dynamics in Iberian red deer. The management of Iberian red deer populations should take into account cohort effects and supplemental feeding practices, which can buffer density- and climate-dependent effects and reduce natural selection pressures.     

The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH,virulence and bacterial survival in plant tissue

We have studied the role of acidic pH as a barrier for the colonization of the plant apoplast by Erwinia chrysanthemi. A minitransposon containing a promoterless reporter gene, gus, was used for random mutagenesis of the bacterial genome. An acid-sensitive mutant, named BT119, was isolated and had the following differential features with respect to the wild-type strain: (i) inability to grow at pH 相似文献   

Both N- and C-terminal domains of parathyroid hormone-related protein increase interleukin-6 by nuclear factor-kappa B activation in osteoblastic cells

Parathyroid hormone (PTH)-related protein (PTHrP) seems to affect bone resorption by interaction with bone cytokines, among them interleukin-6 (IL-6). Recent studies suggest that nuclear factor (NF)-kappaB activation has an important role in bone resorption. We assessed whether the N-terminal fragment of PTHrP, and its C-terminal region, unrelated to PTH, can activate NF-kappaB, and its relationship with IL-6 gene induction in different rat and human osteoblastic cell preparations. Here we present molecular data demonstrating that both PTHrP (1-36) and PTHrP (107-139) activate NF-kappaB, leading to an increase in IL-6 mRNA, in these cells. Using anti-p65 and anti-p50 antibodies, we detected the presence of both proteins in the activated NF-kappaB complex. This effect induced by either the N- or C-terminal PTHrP domain in osteoblastic cells appears to occur by different intracellular mechanisms, involving protein kinase A or intracellular Ca(2+)/protein kinase C activation, respectively. However, the effect of each peptide alone did not increase further when added together. Our findings lend support to the hypothesis that the C-terminal domain of PTHrP, in a manner similar to its N-terminal fragment, might stimulate bone resorption. These studies also provide further insights into the putative role of PTHrP as a modulator of bone remodeling.     

Relationship between bronchopulmonary nematode larvae and relative abundances of Spanish ibex (Capra pyrenaica hispanica) from Castilla-La Mancha, Spain

The excretion of bronchopulmonary nematode infective larvae was evaluated in 160 faecal samples of Spanish ibex (Capra pyrenaica hispanica) collected from 13 populations in Castilla-La Mancha, south-central Spain in September 2003. Intensities and prevalences were compared with pasture availability, abundances of wild and domestic ungulates at both levels, i.e. for populations and for faeces in a two-step procedure. Protostrongylid larvae showed similar infection rates (mean intensity: 1.56+/-0.12, n=94; mean prevalence: 25.62+/-6.86%, n=160) to Dictyocaulus spp. (mean intensity: 1.03+/-0.11, n=48; mean prevalence: 30.00+/-7.11%, n=160). At the population level, positive correlations were found between the prevalences of both bronchopulmonary taxa. The prevalence in both groups, but not intensity, also correlated positively with Spanish ibex abundance indexes both for the populations and individual faeces. These findings suggest that: (i) parasite spreading across Spanish ibex populations in Castilla-La Mancha could respond to host density-dependent processes; and (ii) these populations may have similar exposition and/or susceptibility to both bronchopulmonary taxa resulting in similar host-parasite patterns, despite their different life cycles. Bronchopulmonary outputs in the Spanish ibex from Castilla-La Mancha seems not to represent a health risk for this endemic wild ungulate but may be useful in any health surveillance scheme for the increasing populations of Spanish ibex.     

Risk factors for the detected presence of Mycobacterium bovis in cattle in south central Spain

Tuberculosis (TB) is a chronic bacterial disease of livestock and wildlife, which has major social and economic costs. In Spain, cattle test-and-slaughter schemes have dramatically reduced TB levels, but a wildlife reservoir of the disease is thought to be preventing total eradication. We aim to identify the risk factors for the presence of TB in cattle in Spain. In this case–control study, we combined a farmer-based questionnaire and participatory mapping with government records in Almodovar, Spain. Data were collected from a mixture of TB-free and TB-infected farms, yielding a total sample of 73 farms. Generalised linear modelling and information theory were used to identify the risk factors strongly associated with TB, and farmers were also asked their opinions on TB and wildlife management. The risk factors most strongly associated with TB on a farm were the presence of wildlife, the number of streams per hectare and feeding volume foods (e.g. hay) on the ground. Farmers’ opinions about TB were influenced by their experience of the disease and their interactions with wildlife. The results highlight the complexities of managing TB, and demonstrate the need for a system-level understanding of the inter-relationships among epidemiological, ecological, environmental, social and political risk factors.     

Combined Patterns of IGHV Repertoire and Cytogenetic/Molecular Alterations in Monoclonal B Lymphocytosis versus Chronic Lymphocytic Leukemia

Background

Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBL^hi) or without (MBL^lo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown.

Methodology/Principal Findings

For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBL^lo clones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBL^hi and advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)⁺ clonal B-cells.

Conclusions/Significance

These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBL^lo clones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution.     

Involvement of human choline kinase alpha and beta in carcinogenesis: a different role in lipid metabolism and biological functions

We have summarized here the importance of ChoKα1 in human carcinogenesis. ChoKα1 displays its oncogenic activity through activation of specific signaling pathways that influence on cell proliferation and survival. It is overexpressed in a large number of human tumors with an incidence of 40-60% of all tumors investigated. Currently, there is an active effort in the development of strategies to knockdown the activity of ChoKα through specific siRNA or small molecules inhibitors. Results from genetic silencing or from treatment with MN58b, a well characterized ChoKα inhibitor showing antiproliferative and antitumoral effect in mice xenografts, provide strong support to this concept, indicating that the design of new antitumoral drugs must be selective against this isoform. However, affecting the other two known isoforms of ChoK may have also therapeutic consequences since the physiologically active form of ChoK may be constituted by homo or heterodimers. Furthermore, alteration of the ChoKβ activity might lead to a change in the lipid content of the cells of particular tissues such as skeletal muscle as described in the ChoKβ null mice (Sher et al., 2006). Finally, the identification of the ChoKα1 isoform as an excellent novel tool for the diagnosis and prognosis of cancer patients may have clinical consequences of immediate usefulness. On one hand, the use of specific monoclonal antibodies against ChoKα1 as a tool for diagnosis in paraffin embedded samples from patient biopsies, through standard immunohistochemistry techniques, can now be achieved (Gallego-Ortega et al., 2006). On the other hand, it has been recently described the prognostic value of determination of ChoKα1 expression levels in non-small cell lung cancer using real time quantitative PCR technology (Ramírez de Molina et al., 2007). Therefore, further research should be supported on the utility of ChoK isoforms as a promising area to improve cancer diagnosis and treatment.     

The Iberian ibex is under an expansion trend but displaced to suboptimal habitats by the presence of extensive goat livestock in central Spain

In this paper an updated distribution of the Iberian ibex (Capra pyrenaica, Schinz 1838) in the central Spanish region of Castile–La Mancha is shown. The species is present in 19% of the study region, and in areas not cited so far in the literature. A detailed analysis of habitat suitability was also carried out, applying a␣new methodology, Ecological-Niche Factor Analysis, which uses presence data to␣build a habitat suitability map of a given species. As livestock activity is quite intense in the region, the presence of a potential competitor, the domestic goat (Capra hircus), was included in the analyses. Factors affecting ibex relative abundance were determined by means of a nested stepwise multiple regression, where livestock presence/absence was the nested factor. The presence of livestock has a negative effect on ibex relative abundance, causing the ibex to select areas of poor, sparse vegetation, cultivated lands and forests, whereas in the absence of livestock, the ibex is mainly present in pasture–scrub lands and non-cultivated lands. Conservation implications of these results are discussed in the context of a Mediterranean region where extensive livestock grazing systems abound.     

ATP insertion opposite 8-oxo-deoxyguanosine by Pol4 mediates error-free tolerance in Schizosaccharomyces pombe

7,8-Dihydro-8-oxo-deoxyguanosine (8oxodG) is a highly premutagenic DNA lesion due to its ability to mispair with adenine. Schizosaccharomyces pombe lacks homologs for relevant enzymes that repair 8oxodG, which suggests that this lesion could be persistent and must be tolerated. Here we show that SpPol4, the unique PolX in fission yeast, incorporates ATP opposite 8oxodG almost exclusively when all nucleotides (ribos and deoxys) are provided at physiological concentrations. Remarkably, this SpPol4-specific reaction could also occur during the NHEJ of DSBs. In cell extracts, misincorporation of ATP opposite 8oxodG was shown to be SpPol4-specific, although RNase H2 efficiently recognized the 8oxodG:AMP mispair to remove AMP and trigger error-free incorporation of dCTP. These data are the first evidence that ribonucleotides can be used safely for 8oxodG tolerance, suggesting that insertion of the highly abundant ATP substrate could be beneficial to promote efficient and error-free repair of 8oxodG-associated DSBs. Moreover, we demonstrate that purified SpPol4 uses 8oxo-dGTP and 8oxo-GTP as substrates for DNA polymerization, although with poor efficiency compared to the incorporation of undamaged nucleotides opposite either 8oxodG or undamaged templates. This suggests that SpPol4 is specialized in tolerating 8oxodG as a DNA template, without contributing significantly to the accumulation of this lesion in the DNA.