Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.     

Responses of Nine Trifolium repens L. Populations to Ultraviolet-B Radiation: Differential Flavonol Glycoside Accumulation and Biomass Production

This study aimed to quantify and identify flavonoids involvedin the response of nine populations of white clover (Trifoliumrepens L.) to ultraviolet-B radiation (UV-B). Plants were grownfor 12 weeks in controlled environment rooms with or withoutsupplemental UV-B radiation of 13.3 kJ m^-2d^-1. Methanol–waterextractable flavonoids were quantified using high performanceliquid chromatography (HPLC). Two major peaks showed significantenhancement in the HPLC chromatogram in response to supplementalUV-B. The structures of the compounds responsible were identifiedby¹H and¹³C nuclear magnetic resonance (NMR) spectroscopy tobe the flavonols quercetin-3-O-ß- D -xylopyranosyl-(1 2)-ß- D -galactopyranoside and kaempferol-3-O-ß-D -xylopyranosyl-(1 2)-ß- D -galactopyranoside. Withsupplemental UV-B, quercetin glycoside levels increased on averageby 200% while the kaempferol glycoside response was much smaller.Significant differences in flavonol accumulation were foundamong T. repens populations, both constitutively and in responseto UV-B. Stress-adapted populations displayed particularly highflavonol levels under UV-B. There was an inverse correlationbetween plant productivity and quercetin accumulation. Furthermore,higher quercetin accumulation under UV-B was correlated withtolerance against UV-B-induced growth reduction. In conclusion,within-species comparisons in T. repens lend support to a distinctrole for ortho -dihydroxylated flavonoids in the adaptationto UV-B stress and suggest particular advantages in this UV-B-inducedbiochemical adaptation for populations characterized by lowhabitat and plant productivity. Copyright 2000 Annals of BotanyCompany Ultraviolet-B, Trifolium repens, white clover, HPLC, NMR, flavonoids, flavonols, quercetin, kaempferol, biomass, genetic variation, intraspecific     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999     

Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism

ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd³⁺ caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd³⁺. Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd³⁺, while NO donors rescued apyrase- and Gd³⁺-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd³⁺-sensitive receptor that is coupled with intracellular NO production.     

Comparison of Bayesian models to estimate direct genomic values in multi-breed commercial beef cattle

Background

While several studies have examined the accuracy of direct genomic breeding values (DGV) within and across purebred cattle populations, the accuracy of DGV in crossbred or multi-breed cattle populations has been less well examined. Interest in the use of genomic tools for both selection and management has increased within the hybrid seedstock and commercial cattle sectors and research is needed to determine their efficacy. We predicted DGV for six traits using training populations of various sizes and alternative Bayesian models for a population of 3240 crossbred animals. Our objective was to compare alternate models with different assumptions regarding the distributions of single nucleotide polymorphism (SNP) effects to determine the optimal model for enhancing feasibility of multi-breed DGV prediction for the commercial beef industry.

Results

Realized accuracies ranged from 0.40 to 0.78. Randomly assigning 60 to 70% of animals to training (n ≈ 2000 records) yielded DGV accuracies with the smallest coefficients of variation. Mixture models (BayesB95, BayesCπ) and models that allow SNP effects to be sampled from distributions with unequal variances (BayesA, BayesB95) were advantageous for traits that appear or are known to be influenced by large-effect genes. For other traits, models differed little in prediction accuracy (~0.3 to 0.6%), suggesting that they are mainly controlled by small-effect loci.

Conclusions

The proportion (60 to 70%) of data allocated to training that optimized DGV accuracy and minimized the coefficient of variation of accuracy was similar to large dairy populations. Larger effects were estimated for some SNPs using BayesA and BayesB95 models because they allow unequal SNP variances. This substantially increased DGV accuracy for Warner-Bratzler Shear Force, for which large-effect quantitative trait loci (QTL) are known, while no loss in accuracy was observed for traits that appear to follow the infinitesimal model. Large decreases in accuracy (up to 0.07) occurred when SNPs that presumably tag large-effect QTL were over-regressed towards the mean in BayesC0 analyses. The DGV accuracies achieved here indicate that genomic selection has predictive utility in the commercial beef industry and that using models that reflect the genomic architecture of the trait can have predictive advantages in multi-breed populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0106-8) contains supplementary material, which is available to authorized users.     

Fluvastatin suppresses native and recombinant human P2X4 receptor function

Statins have both cholesterol lowering and anti-inflammatory activities, whether mechanisms underlying their activities are independent remains unclear. The ATP-gated P2X(4) receptor is a pro-inflammatory mediator. Here, we investigate the action of fluvastatin and other cholesterol depleting agents on native and recombinant human P2X(4) receptor. Fluvastatin and mβCD suppressed P2X(4)-dependent calcium influx in THP-1 monocytes, without affecting P2Y receptor responses. mβCD or filipin III suppressed the current density of recombinant human P2X(4) receptors. Human P2X(2) was insensitive to cholesterol depletion. Cholesterol depletion had no effect on intrinsic P2X(4) receptor properties as judged by ATP concentration-response relationship, receptor rundown or current decay during agonist occupancy. These data suggest fluvastatin suppresses P2X(4) activity in monocytes through cholesterol depletion and not by modulating intrinsic channel properties.     

Spatial and Temporal Analysis of Contact Rates in Female White-Tailed Deer

Abstract: White-tailed deer (Odocoileus virginianus) are important game mammals and potential reservoirs of diseases of domestic livestock; thus, diseases of deer are of great concern to wildlife managers. Contact, either direct or indirect, is necessary for disease transmission, but we know little about the ecological contexts that promote intrasexual contact among deer. Using pair-wise direct contacts estimated from Global Positioning System collar locations and joint utilization distributions (JUDs), we assessed habitats in which contacts occur to test whether direct contact rates among female white-tailed deer in different social groups differs among land-cover types. We also tested whether contact rates differed among seasons, lunar phases, and times of day. We obtained locations from 27 female deer for periods of 0.5–17 months during 2002–2006. We designated any simultaneous pair of locations for 2 deer <25 m apart as a direct contact. For each season, we used compositional analysis to compare land-cover types where 2 deer had contact to available land-cover weighted by their JUD. We used mixed-model logistic regression to test for effects of season, lunar phase, and time of day on contact rates. Contact rates during the gestation season were greater than expected from random use in forest and grassland cover, whereas contact rates during the fawning period were greater in agricultural fields than in other land-cover types. Contact rates were greatest during the rut and lowest in summer. Diel patterns of contact rates varied with season, and contact rates were elevated during full moon compared to other lunar periods. Both spatial and temporal analyses suggest that contact between female deer in different social groups occurs mainly during feeding, which highlights the potential impact of food distribution and habitat on contact rates among deer. By using methods to associate contacts and land-cover, we have created beneficial tools for more elaborate and detailed studies of disease transmission. Our methods can offer information necessary to develop spatially realistic models of disease transmission in deer.     

Permeation properties of a P2X receptor in the green algae Ostreococcus tauri

We have cloned a P2X receptor (OtP2X) from the green algae Ostreococcus tauri. The 42-kDa receptor shares approximately 28% identity with human P2X receptors and 23% with the Dictyostelium P2X receptor. ATP application evoked flickery single channel openings in outside-out membrane patches from human embryonic kidney 293 cells expressing OtP2X. Whole-cell recordings showed concentration-dependent cation currents reversing close to zero mV; ATP gave a half-maximal current at 250 mum. alphabeta-Methylene-ATP evoked only small currents in comparison to ATP (EC(50) > 5 mm). 2',3'-O-(4-Benzoylbenzoyl)-ATP, betagamma-imido-ATP, ADP, and several other nucleotide triphosphates did not activate any current. The currents evoked by 300 mum ATP were not inhibited by 100 microm suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, 2',3'-O-(2,4,6-trinitrophenol)-ATP, or copper. Ion substitution experiments indicated permeabilities relative to sodium with the rank order calcium >choline >Tris >tetraethylammonium >N-methyl-D-glucosamine. However, OtP2X had a low relative calcium permeability (P(Ca)/P(Na) = 0.4) in comparison with other P2X receptors. This was due at least in part to the presence of an asparagine residue (Asn(353)) at a position in the second transmembrane domain in place of the aspartate that is completely conserved in all other P2X receptor subunits, because replacement of Asn(353) with aspartate increased calcium permeability by approximately 50%. The results indicate that the ability of ATP to gate cation permeation across membranes exists in cells that diverged in evolutionary terms from animals about 1 billion years ago.     

The CHD3 remodeler PICKLE promotes trimethylation of histone H3 lysine 27

CHD3 proteins are ATP-dependent chromatin remodelers that contribute to repression of developmentally regulated genes in both animal and plant systems. In animals, this repression has been linked to a multiple subunit complex, Mi-2/NuRD, whose constituents include a CHD3 protein, a histone deacetylase, and a methyl-CpG-binding domain protein. In Arabidopsis, PICKLE (PKL) codes for a CHD3 protein that acts during germination to repress expression of seed-associated genes. Repression of seed-associated traits is promoted in pkl seedlings by the plant growth regulator gibberellin (GA). We undertook a microarray analysis to determine how PKL and GA act to promote the transition from seed to seedling. We found that PKL and GA act in separate pathways to repress expression of seed-specific genes. Comparison of genomic datasets revealed that PKL-dependent genes are enriched for trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation studies demonstrate that PKL promotes H3K27me3 in both germinating seedlings and in adult plants but do not identify a connection between PKL-dependent expression and acetylation levels. Taken together, our analyses illuminate a new pathway by which CHD3 remodelers contribute to repression in eukaryotes.