A novel cell array technique for high-throughput,cell-based analysis

Summary Microarray technology has burgeoned over the past few years from nucleic acid-based arrays to tissue microarrays (TMAs). This study aimed to develop a technique to incorporate cell lines into an array and to demonstrate the usefulness of this technique by performing immunohistochemistry for β-catenin. Cell suspensions were prepared from 23 tumor cell lines. These were fixed in formalin, suspended in agar, and embedded in paraffin to produce a cell block. A “tissue microarrayer” was used to remove triplicate, 0.6 mm-cores from each cell block and to transfer these into a recipient paraffin block at precise coordinates. Immunohistochemistry was used to identify cell lines positive for β-catenin. Cultured cells were successfully incorporated into the microarray, with preservation of cell architecture and even distribution of cells within each core. A total of 18 of 69 cores (26%) were lost in processing. A total of 16 of 23 cell lines were identified as positive for membrane and cytoplasmic β-catenin, and 6 of 23 were negative. Only one cell line was unscorable because of complete core loss. We have developed a “cell microarray” technique for analyzing antigen expression by immunohistochemistry in multiple cell lines in a single expriment. This novel application of microarrays permits high-throughput, cost-efficient analysis, with the potential to rapidly identify markers with potential diagnostic and therapeutic implications in human disease.     

Multiple QTLs influencing triglyceride and HDL and total cholesterol levels identified in families with atherogenic dyslipidemia

We conducted a genome-wide scan using variance components linkage analysis to localize quantitative-trait loci (QTLs) influencing triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol, and total cholesterol (TC) levels in 3,071 subjects from 459 families with atherogenic dyslipidemia. The most significant evidence for linkage to TG levels was found in a subset of Turkish families at 11q22 [logarithm of the odds ratio (LOD)=3.34] and at 17q12 (LOD=3.44). We performed sequential oligogenic linkage analysis to examine whether multiple QTLs jointly influence TG levels in the Turkish families. These analyses revealed loci at 20q13 that showed strong epistatic effects with 11q22 (conditional LOD=3.15) and at 7q36 that showed strong epistatic effects with 17q12 (conditional LOD=3.21). We also found linkage on the 8p21 region for TG in the entire group of families (LOD=3.08). For HDL-C levels, evidence of linkage was identified on chromosome 15 in the Turkish families (LOD=3.05) and on chromosome 5 in the entire group of families (LOD=2.83). Linkage to QTLs for TC was found at 8p23 in the entire group of families (LOD=4.05) and at 5q13 in a subset of Turkish and Mediterranean families (LOD=3.72). These QTLs provide important clues for the further investigation of genes responsible for these complex lipid phenotypes. These data also indicate that a large proportion of the variance of TG levels in the Turkish population is explained by the interaction of multiple genetic loci.     

Disruption of the Arabidopsis AtKu80 gene demonstrates an essential role for AtKu80 protein in efficient repair of DNA double-strand breaks in vivo

Double-strand breaks (DSBs) in DNA may occur spontaneously in the cell or be induced experimentally by gamma-irradiation, and represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination appears to be the major pathway for DSB repair in humans and plants, and it may also be the major route whereby T-DNA integrates into the plant genome during cell transformation. In yeast and mammals, the exposed ends of damaged DNA are bound with high affinity by a dimer of Ku70 and Ku80 proteins, which protects the ends from exonucleases and juxtaposes the two ends of the DSB, independent of sequence homology. Here we report the functional characterization of Ku70 and Ku80 from Arabidopsis thaliana, and demonstrate that AtKu80 and AtKu70 form a heterodimer with DNA binding activity that is specific for DNA ends. An atku80 knockout mutant shows hypersensitivity to the DNA-damaging agents menadione and bleomycin, consistent with a role for AtKu80 in the repair of DSBs in vivo in Arabidopsis.     

Plasma levels of remnant particles are determined in part by variation in the APOC3 gene insulin response element and the APOCI-APOE cluster

Remnant particles of triglyceride-rich lipoproteins (RLP) are known to be a strong predictor of atherogenicity. The serum concentrations of remnant-like particle triglyceride (RLPTG) and remnant-like particle cholesterol (RLPC) have been determined in a representative sample of the Czech MONICA study (n = 285). The relationship was investigated between remnant particle triglyceride/cholesterol concentrations and polymorphisms in the genes APOC3 (-482C-->T/3238C-->G), APOE (epsilon2/epsilon3/epsilon4), APOCI (-317-321ins), APOB (signal peptide), hepatic lipase (LIPE, -480C-->T), and lipoprotein lipase (LPL, S447X). Univariate analysis showed significant effects on RLPTG associated only with the APOE genotype (P = 0.009), the APOC3 -482C-->T genotype (P = 0.018), and the APOCI -317-321ins (P = 0.014) genotype and significant effects on RLPC with APOE (P = 0.01) and APOCI -317-321ins (P = 0.021). The raising effect of the APOE genotype for both remnant cholesterol and triglyceride was confined to the epsilon2/4 (n = 6) and varepsilon4/4 (n = 3) groups, and thus when the epsilon2/4 group was omitted in order to analyze by allele (epsilon2+/epsilon3+/epsilon4+), significance was lost (P = 0.6). There was strong linkage disequilibrium between the APOE and APOCI alleles (chi(2), P < 0.001) and a multivariate ANOVA of RLPTG with all three significantly associated variants as factors demonstrated that while the APOC3 -482C-->T effect was independent of the others (P = 0.003), the APOCI -317-321ins and APOE effects were not. This was also true for the APOCI -317-321ins and APOE effects on RLPC. To assess whether APOE-CI effects on RLPC were independent of their effects on total cholesterol and triglyceride levels, multiple linear regression was used. Using multiple linear regression, it appeared that the APOE-CI effects on RLPC were independent of their effects on plasma cholesterol, but the effects of APOC3 and APOE-CI on RLPTG could not be separated from their effects on plasma Tg levels.Further characterization of this remnant particle phenotype and its genetic determinants may lead to a better understanding of its metabolism and contribution to atherosclerosis.     

Arabidopsis DNA ligase IV is induced by gamma-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4

Rejoining of single- and double-strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double-strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by gamma-irradiation, represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP-dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino-acid sequence to the human DNA ligase IV. Yeast two-hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV-binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C-terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by gamma-irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.     

静磁场对单株人体体表正常菌生长影响的研究

本文通过40mT和120mT两种静磁场作用下表皮葡萄球菌生长过程的研究,发现试验所选强度静磁场加速了表皮葡萄球菌在对数生长期的生长速率,而在进入稳定生长期后其生长速率反而低于对照组,但就整个生长周期而言,静磁场作用下表皮葡萄球菌的总量大于对照组,表明了试验所选静磁场对表皮葡萄球菌生长有一定促进作用.     

Testing the generality of above‐ground biomass allometry across plant functional types at the continent scale

Accurate ground‐based estimation of the carbon stored in terrestrial ecosystems is critical to quantifying the global carbon budget. Allometric models provide cost‐effective methods for biomass prediction. But do such models vary with ecoregion or plant functional type? We compiled 15 054 measurements of individual tree or shrub biomass from across Australia to examine the generality of allometric models for above‐ground biomass prediction. This provided a robust case study because Australia includes ecoregions ranging from arid shrublands to tropical rainforests, and has a rich history of biomass research, particularly in planted forests. Regardless of ecoregion, for five broad categories of plant functional type (shrubs; multistemmed trees; trees of the genus Eucalyptus and closely related genera; other trees of high wood density; and other trees of low wood density), relationships between biomass and stem diameter were generic. Simple power‐law models explained 84–95% of the variation in biomass, with little improvement in model performance when other plant variables (height, bole wood density), or site characteristics (climate, age, management) were included. Predictions of stand‐based biomass from allometric models of varying levels of generalization (species‐specific, plant functional type) were validated using whole‐plot harvest data from 17 contrasting stands (range: 9–356 Mg ha^?1). Losses in efficiency of prediction were <1% if generalized models were used in place of species‐specific models. Furthermore, application of generalized multispecies models did not introduce significant bias in biomass prediction in 92% of the 53 species tested. Further, overall efficiency of stand‐level biomass prediction was 99%, with a mean absolute prediction error of only 13%. Hence, for cost‐effective prediction of biomass across a wide range of stands, we recommend use of generic allometric models based on plant functional types. Development of new species‐specific models is only warranted when gains in accuracy of stand‐based predictions are relatively high (e.g. high‐value monocultures).     

Probable Site of Sex Pheromone Emission in Female Vine and Obscure Mealybugs (Hemiptera: Pseudococcidae)

In laboratory bioassays, male Pseudococcus viburni (Signoret) and Planococcus ficus Signoret were attracted to body sections of females that included the metathoracic legs. Extracts of the metathoracic legs of female P. ficus were much more attractive to males than extracts of the other legs, and males exhibited copulatory behavior towards these extracts. Scanning electron microscopy showed the presence of translucent pores on the coxae of mature but not immature females. Collectively, the data suggest that the female-produced sex pheromones of these species are released from the metathoracic legs, probably from the translucent pores.