Disruption of mechanisms that prevent rereplication triggers a DNA damage response

Eukaryotes replicate DNA once and only once per cell cycle due to multiple, partially overlapping mechanisms efficiently preventing reinitiation. The consequences of reinitiation are unknown. Here we show that the induction of rereplication by mutations in components of the prereplicative complex (origin recognition complex [ORC], Cdc6, and minichromosome maintenance proteins) causes a cell cycle arrest with activated Rad53, a large-budded morphology, and an undivided nucleus. Combining a mutation disrupting the Clb5-Orc6 interaction (ORC6-rxl) and a mutation stabilizing Cdc6 (CDC6(Delta)NT) causes a cell cycle delay with a similar phenotype, although this background is only partially compromised for rereplication control and does not exhibit overreplication detectable by fluorescence-activated cell sorting. We conducted a systematic screen that identified genetic requirements for the viability of these cells. ORC6-rxl CDC6(Delta)NT cells depend heavily on genes required for the DNA damage response and for double-strand-break repair by homologous recombination. Our results implicate an Mre11-Mec1-dependent pathway in limiting the extent of rereplication.     

Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.     

ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.     

Fatal pulmonary acariasis in an aged indoor rhesus macaque (Macaca mulatta)

Pulmonary acariasis is a sporadic, incidental finding in colony‐raised rhesus macaques (Macaca mulatta). Prophylactic treatment in indoor‐raised and indoor‐housed macaques is not routine due to low prevalence, lack of clinical significance, and potential risk of toxicosis. This case is an unusually severe infestation of Pneumonyssus simicola in an indoor‐housed rhesus macaque, which ultimately resulted in this animal's death.     

Temporal dynamics and relationship between climate,limnological variables and zooplankton composition in climate-sensitive Lake Bosumtwi,Ghana

The community composition and the factors affecting seasonal and interannual dynamics of zooplankton in Lake Bosumtwi were studied biweekly at a central index station during 2005 and 2006. The lake zooplankton community was species poor. Mesocyclops bosumtwii was numerically superior seasonally and interannually and was endemic to the lake. Minor constituents included Moina micrura, six rotifer species (except for Hexarthra intermedia) and Chaoborus ceratopogones larvae. Low variance of cyanobacteria-dominated phytoplankton biomass underlined stable zooplankton community structure. Emergence of rare species of rotifers occurred seasonally. The climatic signature on the lake’s stratification and mixing regime was strongly influenced by atmospheric temperature, but weakly by wind strength, because of sheltering of the lake by high crater walls. Increasing mixing depth entrained high TP concentrations from below the thermocline seasonally, but reflected poorly in the phytoplankton biomass behaviour. Total zooplankton abundance did not differ seasonally, but varied markedly from year to year in its timing and magnitude. Herbivores were squeezed between food limitation and high predation pressure from Chaoborus all year round. The low fish planktivory (high fishing pressure) on Chaoborus may create a trophic bottleneck restricting energy transfer efficiency from zooplankton to fish.     

The p400 complex is an essential E1A transformation target

Here, we report the identification of a new E1A binding protein complex that is essential for E1A-mediated transformation. Its core component is a SWI2/SNF2-related, 400 kDa protein (p400). Other components include the myc- and p/CAF-associated cofactor, TRRAP/PAF400, the DNA helicases TAP54alpha/beta, actin-like proteins, and the human homolog of the Drosophila Enhancer of Polycomb protein. An E1A mutant, defective in p400 binding, is also defective in transformation. Certain p400 fragments partially rescued this phenotype, underscoring the role of E1A-p400 complex formation in the E1A transforming process. Furthermore, E1A and c-myc each alter the subunit composition of p400 complexes, implying that physiological p400 complex formation contributes to transformation suppression.     

Implications phylogénétiques de I'ultrastructure de la spermatogenèse, du spermatozoïde et de I'ovogenèse du TurbellariéUrastoma cyprinae ("Prolecithophora", Urastomidae)

Spermiogenesis in Urastoma cyprinae (Graff. 1882) involvcs a progressive lengthening of the spermatid. Free flagella are only transitory. The mature spermatozoon is fusibrm. 45μm in length and 2.5 μm in width. It contains two incorporated axonermes of the flatworm 9+"1" pattern, two elongated mitochondria. an elongated nucleus and a row or cortical longitudinal microtubules. Observations on oogenesis concerncd only the immuturc ovary. lmmature oocytes contain few dense granules and accssory cells were not ohserved. Phylogenetic implications of a biflagellate spermatozoon in a Prolecithophora are important. The presence of two 9 +"1" axonemes confirms that Urastoma (and the Prolecithophora) belongs to the taxon Trepaxonemata Ehlers. Previous electron microscope studies on spermitozoa of Prolecithophora (four genera) only dealt with aflagellate spermatozoa. On this basis, Ehlers (1985) proposed two autapomorphics for the taxon Prolecithophora: aflagellate spermatozoon and spermatozoal mitochondrial derivatives with abundant membranes. The present observations on Urastoma contradict these two autapomorphics. The taxon Prolecithophora cannot be defined by autapomorphies of the spermatozoon.     

Genome wide DNA copy number analysis of serous type ovarian carcinomas identifies genetic markers predictive of clinical outcome

Ovarian cancer is the fifth leading cause of cancer death in women. Ovarian cancers display a high degree of complex genetic alterations involving many oncogenes and tumor suppressor genes. Analysis of the association between genetic alterations and clinical endpoints such as survival will lead to improved patient management via genetic stratification of patients into clinically relevant subgroups. In this study, we aim to define subgroups of high-grade serous ovarian carcinomas that differ with respect to prognosis and overall survival. Genome-wide DNA copy number alterations (CNAs) were measured in 72 clinically annotated, high-grade serous tumors using high-resolution oligonucleotide arrays. Two clinically annotated, independent cohorts were used for validation. Unsupervised hierarchical clustering of copy number data derived from the 72 patient cohort resulted in two clusters with significant difference in progression free survival (PFS) and a marginal difference in overall survival (OS). GISTIC analysis of the two clusters identified altered regions unique to each cluster. Supervised clustering of two independent large cohorts of high-grade serous tumors using the classification scheme derived from the two initial clusters validated our results and identified 8 genomic regions that are distinctly different among the subgroups. These 8 regions map to 8p21.3, 8p23.2, 12p12.1, 17p11.2, 17p12, 19q12, 20q11.21 and 20q13.12; and harbor potential oncogenes and tumor suppressor genes that are likely to be involved in the pathogenesis of ovarian carcinoma. We have identified a set of genetic alterations that could be used for stratification of high-grade serous tumors into clinically relevant treatment subgroups.     

Stoichiometric relationships in vernal pond plankton communities

1. The light-nutrient hypothesis (LNH) predicts that changes in light supply can alter the balance of nutrient and energy limitation in primary producers. We tested this prediction by examining temporal changes in vernal forest ponds, which are highly dynamic systems with respect to seasonal change in light and nutrient supply. In three vernal ponds that differ in productivity, we measured changes in light, total and seston nitrogen and phosphorus, and seston carbon and chlorophyll during the spring, before and after tree leaf-out. We also quantified changes in the population dynamics of the major zooplankton grazers in these systems.
2. In each pond, nutrient levels increased and light levels declined, creating a temporal shift in light-nutrient supply to the plankton. Results generally supported predictions of stoichiometric theory and the LNH, but there were notable exceptions.
3. Seston C : N : P ratios rapidly changed in response to dramatic increases in N and P supply rates. However, seston N : P was typically lower than values for total N : P in the water. Furthermore, as predicted, we observed a decline in seston C : P as the light : nutrient ratio declined, but seston C : N simultaneously increased. These results suggest an unexpected shift towards potential nitrogen limitation. Alternatively, this change in nutrient ratios may be driven by a seasonal change in phytoplankton composition or nutritional mode.
4. Seston carbon concentrations remained stable despite seasonal changes in grazing intensity associated with the phenology of large-bodied Daphnia grazers. However, chlorophyll concentrations declined dramatically as the season progressed, resulting in a simultaneous decline in the C : Chlorophyll ratio of seston. Both pond shading and increased grazing probably contributed to the decline in chlorophyll.