Regulation of DNA Double Strand Break Response

To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.

The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.

Appropriateness of Largemouth Bass as a Model Species for Detection of Endocrine Dissruption

Intersex in largemouth bass (Micropterus salmoides) has been correlated with regional anthropogenic activity, but has not been causally linked to environmental factors. Four groups of hatchery-reared largemouth bass (LMB) and fathead minnows (FHM) of varying ages and sex were exposed to aqueous poultry litter mixtures, 17β- estradiol (E2), and controls. Water samples were analyzed for estrogens through liquid chromatography tandem mass spectrometry and estrogenicity through the bioluminescent yeast estrogen screen assay. Fish plasma was analyzed for the egg yolk protein vitellogenin (Vtg) using enzyme–linked immunosorbent assay and gonad tissue was examined histologically for enumeration of testicular oocytes (TO). Water chemistry revealed typical E2 conversion to Estrone with subsequent decay over the exposure periods. A modest prevalence of TO (9.4%) was detected with no apparent treatment effect. While significant Vtg induction was found in E2 exposed FHM, minimal Vtg induction was found in male LMB. Despite field findings of intersex in male LMB, this species may be poorly suited for laboratory investigations into endocrine disruption.

Basophil activation tests for the diagnosis of food allergy in children.

BACKGROUND: Positive skin prick tests (SPT) for food allergens and specific IgE (sIgE) in serum indicate sensitization but do not enable distinction between sensitized but tolerant and clinically allergic patients. OBJECTIVE: Herein, we evaluate the clinical relevance of basophil activation tests (BATs) for peanut or egg allergy diagnosis. METHODS: Thirty-two peanut-allergic, 14 peanut-sensitized (sIgE(+) and/or SPT(+) to peanuts) but tolerant children and 29 controls with no history of an adverse reaction to peanuts were included. Similarly, 31 egg-allergic, 14 egg-sensitized children (sIgE(+) and/or SPT(+) to egg white) and 22 controls were studied. Flow cytometric analysis of CD63 expression or CD203c upregulation on basophils and the production of leukotrienes (LT) were performed in response to an in vitro crude peanut extract or ovalbumin (OVA) challenge. RESULTS: After in vitro peanut challenge, the basophils from peanut-allergic children showed significantly higher levels of activation than those from controls (P<0.001). After OVA challenge, a similar distinction (P<0.001) was observed between egg-allergics and controls. Interestingly, the majority of egg- or peanut-sensitized children failed to activate basophils, respectively, in response to OVA and peanut challenge. The sensitivity of the CD63, CD203c and LT assay was 86.7%, 89.5% and 76.0% with a specificity of 94.1%, 97.1% and 94.6% for peanut allergy diagnosis. The corresponding performances of BATs applied to egg allergy diagnosis were 88.9%, 62.5% and 77.8% for the sensitivity and 100%, 96.4% and 96.4% for the specificity. CONCLUSION: Neither conventional tests nor BATs are sensitive and specific enough to predict food allergy accurately. However, BATs may helpfully complete conventional tests, especially SPT, allowing improved discrimination between allergic and non-allergic individuals.

Presencia de Anagrus flaveolus en la Argentina parasitoide de un insecto dañino del trigo y maíz (Insecta - Hymenoptera - Mymaridae)

p.19-23

Experiments and observations made at the Plymouth Laboratory

Diagnostic characters in flat fishes. Development of the egg in flat fishes & pipe fishes. A piebald plaice. Growth & distribution of young food-fishes. Notes on rare or interesting specimens.

Use of satellite data for modelling food availability and survival of marine fish larvae

A modelling scheme is described which uses satellite retrieved sea-surface temperature and chlorophyll-a to derive monthly zooplankton biomass estimates in the eastern North Atlantic; this forms part of a bio-physical model of inter-annual variations in the growth and survival of larvae and post-larvae of mackerel (Scomber scombrus). The temperature and chlorophyll data are incorporated first to model copepod (Calanus) egg production rates. Egg production is then converted to available food using distribution data from the Continuous Plankton Recorder (CPR) Survey, observed population biomass per unit daily egg production and the proportion of the larval mackerel diet comprising Calanus. Results are validated in comparison with field observations of zooplankton biomass. The principal benefit of the modelling scheme is the ability to use the combination of broad scale coverage and fine scale temporal and spatial variability of satellite data as driving forces in the model; weaknesses are the simplicity of the egg production model and the broad-scale generalizations assumed in the raising factors to convert egg production to biomass.

Distribution and abundance of sardine (Sardina pilchardus) eggs in the English Channel from Continuous Plankton Recorder sampling

Continuous Plankton Recorder (CPR) samples from the English Channel and adjacent Celtic shelf, taken over the period 1958-1980, were analysed for sardine (Sardina pilchardus) eggs. Results showed the progression of sardine spawning along the English Channel from west to east from March to August and a return from east to west from September to November. This corresponds with the two seasonal peaks of sardine egg abundance in the western Channel: the main summer peak being in May/June, with a smaller autumn peak in October/November. Long-term changes in sardine egg abundance in CPR samples showed a decline in summer spawning from the late 1960s, but no clear trend in autumn-spawned egg abundance. Similar patterns were observed in the numbers of sardine eggs sampled by conventional plankton net tows at the time-series Station L5 off Plymouth. This supports the use of the longer time-series of sardine egg data at L5 as being representative of a wider area and emphasizes the importance in continuation of the L5 time-series.

Simulation of mackerel (Scomber scombrus) recruitment with an individual-based model and comparison with field data

An individual-based model (IBM) for the simulation of year-to-year survival during the early life-history stages of the north-east Atlantic stock of mackerel (Scomber scombrus) was developed within the EU funded Shelf-Edge Advection, Mortality and Recruitment (SEAMAR) programme. The IBM included transport, growth and survival and was used to track the passive movement of mackerel eggs, larvae and post-larvae and determine their distribution and abundance after approximately 2 months of drift. One of the main outputs from the IBM, namely distributions and numbers of surviving post-larvae, are compared with field data as recruit (age-0/age-1 juveniles) distribution and abundance for the years 1998, 1999 and 2000. The juvenile distributions show more inter-annual and spatial variability than the modelled distributions of survivors; this may be due to the restriction of using the same initial egg distribution for all 3 yr of simulation. The IBM simulations indicate two main recruitment areas for the north-east Atlantic stock of mackerel, these being Porcupine Bank and the south-eastern Bay of Biscay. These areas correspond to areas of high juvenile catches, although the juveniles generally have a more widespread distribution than the model simulations. The best agreement between modelled data and field data for distribution (juveniles and model survivors) is for the year 1998. The juvenile catches in different representative nursery areas are totalled to give a field abundance index (FAI). This index is compared with a model survivor index (MSI) which is calculated from the total of survivors for the whole spawning season. The MSI compares favourably with the FAI for 1998 and 1999 but not for 2000; in this year, juvenile catches dropped sharply compared with the previous years but there was no equivalent drop in modelled survivors.

Modelling the vertical distribution of eggs of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus)

Measurements were made of the density and settling velocity of eggs of sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus), using a density-gradient column. These results were related to observed vertical distributions of eggs obtained from stratified vertical distribution sampling in the Bay of Biscay. Eggs of both species had slightly positive buoyancy in local seawater throughout most of their development until near hatching, when there was a marked increase in density and they became negatively buoyant. The settling velocity of anchovy eggs, which are shaped as prolate ellipsoids, was close to predictions for spherical particles of equivalent volume. An improved model was developed for prediction of the settling velocity of sardine eggs, which are spherical with a relatively large perivitelline volume; this incorporated permeability of the chorion and adjustment of the density of the perivitelline fluid to ambient seawater. Eggs of both species were located mostly in the top 20 m of the water column, in increasing abundance towards the surface. A sub-surface peak of egg abundance was sometimes observed at the pycnocline, particularly where this was pronounced and associated with a low-salinity surface layer. There was a progressive deepening of the depth distributions for successive stages of egg development. Results from this study can be applied for improved plankton sampling of sardine and anchovy eggs and in modelling studies of their vertical distribution.