A new look towards BAC-based array CGH through a comprehensive comparison with oligo-based array CGH

Background

Natural populations of the teleost fish Fundulus heteroclitus tolerate a broad range of environmental conditions including temperature, salinity, hypoxia and chemical pollutants. Strikingly, populations of Fundulus inhabit and have adapted to highly polluted Superfund sites that are contaminated with persistent toxic chemicals. These natural populations provide a foundation to discover critical gene pathways that have evolved in a complex natural environment in response to environmental stressors.

Results

We used Fundulus cDNA arrays to compare metabolic gene expression patterns in the brains of individuals among nine populations: three independent, polluted Superfund populations and two genetically similar, reference populations for each Superfund population. We found that up to 17% of metabolic genes have evolved adaptive changes in gene expression in these Superfund populations. Among these genes, two (1.2%) show a conserved response among three polluted populations, suggesting common, independently evolved mechanisms for adaptation to environmental pollution in these natural populations.

Conclusion

Significant differences among individuals between polluted and reference populations, statistical analyses indicating shared adaptive changes among the Superfund populations, and lack of reduction in gene expression variation suggest that common mechanisms of adaptive resistance to anthropogenic pollutants have evolved independently in multiple Fundulus populations. Among three independent, Superfund populations, two genes have a common response indicating that high selective pressures may favor specific responses.     

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.     

Molecular Cloning, Genomic Organization, and Expression of a Testicular Isoform of Hormone-Sensitive Lipase

By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSL_tes. Due to an addition of amino acids at the NH₂-termini, rat and human HSL_tesconsist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSL_adi). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSL_adi. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSL_adisequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M_r≈ 120,000) that exhibited catalytic activity similar to that of HSL_adi. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells.     

Glypican (heparan sulfate proteoglycan) is palmitoylated, deglycanated and reglycanated during recycling in skin fibroblasts

Skin fibroblasts treated with brefeldin A produce a recyclingvariant of glypican (a glycosyiphosphatidylinositol-anchoredheparan-sulfate proteoglycan) that is resistant to inositol-specificphospholipase C and incorporates sulfate and glucosamine intoheparan sulfate chains (Fransson, L.-Å. et al., Glycobiology,5, 407–415, 1995). We have now investigated structuralmodifications of recycling glypican, such as fatty acylationfrom [³H]palmitate, and degradation and assembly of heparansulfate side chains. Most of the ³H-radioactivity was recoveredas lipid-like material after de-esterification. To distinguishbetween formation of heparan sulfate at vacant sites, elongationof existing chains or degradation followed by re-elongationof chain remnants, cells were pulse-labeled with [³H]glucosamineand then chase-labeled with [¹⁴C]glucosamine. Material isolatedfrom the cells during the chase consisted of proteoglycan andmostly [³H]-labeled heparan-sulfate degradation products (molecularmass, 20–80 kDa) showing that the side chains were degradedduring recycling. The degradation products were initially glucuronate-rich,but became more iduronate-rich with time. The glypican proteoglycanformed during the chase was degraded either with alkali to releaseintact side chains or with heparinase to generate distally locatedchain fragments that were separated from the core protein, containingthe proximally located, covalently attached chain remnants.All of the [¹⁴C]-radioactivity incorporated during the pulsewas found in peripheral chain fragments, and the chains formedwere not significantly longer than the original ones. We thereforeconclude that newly made heparan-sulfate chains were neithermade on vacant sites, nor by extension of existing chains butrather by re-elongation of degraded chain remnants. The remodeledchains made during recycling appeared to be more extensivelymodified than the original ones. fatty acylation glypican heparan sulfate recycling reglycanation     

Chemical competition in target radical reactions: numerical simulation of the theory and comparison with measured oxygen effect on DNA damage in cells

An intracellular radiation-chemical reaction scheme is tested in which solute and solvent radicals R. react with non-target molecules Sa (scavengers) or with target molecules (presumed to be DNA) to produce target radicals T., which may also be produced by direct ionization of DNA. The rate of target radical decomposition to become 'uncommitted damage' that the cell may repair is affected by the concentration of oxygen (O2), thiols (S) and electronaffinic sensitizers (F), which compete with one another to form, respectively, target products TO2, TS and TF. This uncommitted damage is then subject to biochemical modification, including molecular repair, by the cell. The rate equations for this competing reaction scheme were written and programmed for computer simulations of changes in oxygen, thiol and electronaffinic sensitizer concentrations. A reaction scheme that also includes some non-radical target damage was also simulated. Simulations were made using available experimental data concerning intranuclear concentrations and reaction rate constants, respectively, ko, ks and k1 for the reactions T. + O2----TO2, T. + S----TS and T. + F----TF, which produce uncommitted chemical damage. Experimental data on strand-break induction in glutathione-proficient and glutathione-deficient cells, in cells treated with thiol active agents, and in cells treated with hypoxic sensitizers, along with the computer simulations, generally agree that thiol molecules can react with target radicals to reverse T. in competition with O2 and/or electronaffinic sensitizers. Forward reaction rate constants ko, ks (dithiothreitol), ks (glutathione) and k1 (misonidazole) in the approximate ratio 10:0.3:0.02:0.4 satisfied the above reaction scheme, and approximately 5 per cent non-radical target molecule damage could be included with satisfactory agreement with experimental data.     

A novel strategy for microarray quality control using Bayesian networks

MOTIVATION: High-throughput microarray technologies enable measurements of the expression levels of thousands of genes in parallel. However, microarray printing, hybridization and washing may create substantial variability in the quality of the data. As erroneous measurements may have a drastic impact on the results by disturbing the normalization schemes and by introducing expression patterns that lead to incorrect conclusions, it is crucial to discard low quality observations in the early phases of a microarray experiment. A typical microarray experiment consists of tens of thousands of spots on a microarray, making manual extraction of poor quality spots impossible. Thus, there is a need for a reliable and general microarray spot quality control strategy. RESULTS: We suggest a novel strategy for spot quality control by using Bayesian networks, which contain many appealing properties in the spot quality control context. We illustrate how a non-linear least squares based Gaussian fitting procedure can be used in order to extract features for a spot on a microarray. The features we used in this study are: spot intensity, size of the spot, roundness of the spot, alignment error, background intensity, background noise, and bleeding. We conclude that Bayesian networks are a reliable and useful model for microarray spot quality assessment. SUPPLEMENTARY INFORMATION: http://sigwww.cs.tut.fi/TICSP/SpotQuality/.     

The effects of luteinizing hormone releasing hormone (LH-RH) in pregnant rats. I. Postnidatory effects.

The effects of LH-RH on pregnancy in rats were investigated. A single 500 mcg injection of LH-RH on Days 9, 10, or 11 of pregnancy terminated pregnancy, whereas injection on Days 6-8 or 13-16 had little or no effect. The ED 50 on Day 10 for b.i.d. administration was 150 mcg and 550 mcg for a single injection. Administration on Day 9 was followed by a decrease in circulating progesterone levels on Days 10 and 11. The administration of large doses of progesterone reversed the effects of LH-RH administration on Days 7-12. Treatment with estradiol-17beta did not potentiate the effect of progesterone, but appeared to slightly retard fetal resorption when administered alone. The results suggest that the antifertility effect of LH-RH is mediated via functional luteolysis.     

The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function

Many gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated β1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.