Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells

Background

Genomic resources for the majority of free-living vertebrates of ecological and evolutionary importance are scarce. Therefore, linkage maps with high-density genome coverage are needed for progress in genomics of wild species. The Siberian jay (Perisoreus infaustus; Corvidae) is a passerine bird which has been subject to lots of research in the areas of ecology and evolutionary biology. Knowledge of its genome structure and organization is required to advance our understanding of the genetic basis of ecologically important traits in this species, as well as to provide insights into avian genome evolution.

Results

We describe the first genetic linkage map of Siberian jay constructed using 117 microsatellites and a mapping pedigree of 349 animals representing five families from a natural population breeding in western Finland from the years 1975 to 2006. Markers were resolved into nine autosomal and a Z-chromosome-specific linkage group, 10 markers remaining unlinked. The best-position map with the most likely positions of all significantly linked loci had a total sex-average size of 862.8 cM, with an average interval distance of 9.69 cM. The female map covered 988.4 cM, whereas the male map covered only 774 cM. The Z-chromosome linkage group comprised six markers, three pseudoautosomal and three sex-specific loci, and spanned 10.6 cM in females and 48.9 cM in males. Eighty-one of the mapped loci could be ordered on a framework map with odds of >1000:1 covering a total size of 809.6 cM in females and 694.2 cM in males. Significant sex specific distortions towards reduced male recombination rates were revealed in the entire best-position map as well as within two autosomal linkage groups. Comparative mapping between Siberian jay and chicken anchored 22 homologous loci on 6 different linkage groups corresponding to chicken chromosomes Gga1, 2, 3, 4, 5, and Z. Quite a few cases of intra-chromosomal rearrangements within the autosomes and three cases of inter-chromosomal rearrangement between the Siberian jay autosomal linkage groups (LG1, LG2 and LG3) and the chicken sex chromosome GgaZ were observed, suggesting a conserved synteny, but changes in marker order, within autosomes during about 100 million years of avian evolution.

Conclusion

The constructed linkage map represents a valuable resource for intraspecific genomics of Siberian jay, as well as for avian comparative genomic studies. Apart from providing novel insights into sex-specific recombination rates and patterns, the described maps – from a previously genomically uncharacterized superfamily (Corvidae) of passerine birds – provide new insights into avian genome evolution. In combination with high-resolution data on quantitative trait variability from the study population, they also provide a foundation for QTL-mapping studies.     

Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polβDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polβDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polβDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H₂O₂ treatment, polβDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H₂O₂, these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polβDN induced increase in residual γH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual γH2AX foci, indicating an escape from γH2AX-mediated DSB repair. In addition, we show that in the polβDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.     

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.     

The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

Barrier characteristics of brain endothelial cells forming the blood–brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.     

MicroRNAs Regulate Human Adipocyte Lipolysis: Effects of miR-145 Are Linked to TNF-α

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.     

The Helix Rearrangement in the Periplasmic Domain of the Flagellar Stator B Subunit Activates Peptidoglycan Binding and Ion Influx

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The monoclonal antibody GB 42 — a useful marker for the differentiation of myofibroblasts

The expression patterns of a variety of cytoskeletal antigens were studied in normal human tissues (placenta, umbilical cord, myometrium, colon, mammary gland, testis, skeletal muscle, myocardium) as well as in abnormal human tissues (palmar fibromatosis, fibrocystic disease of the mammary gland, mammary carcinoma). The immunohistochemical binding patterns of the monoclonal antibody GB 42 were compared to those of commerical antibodies directed against vimentin, desmin, smooth muscle myosin, pan actin, -smooth muscle actin and -smooth muscle actin. Methods applied comprised immunohistochemistry on cryostat sections and paraffin sections. Immunogold immunocytochemistry was performed on Lowicryl sections. The patterns of GB 42-binding were confirmed biochemically by SDS-PAGE and Western-blotting, and quantitative amino acid analysis. Our data suggest that the monoclonal antibody GB 42 recognizes an actin isoform which is identical to, or closely related to, -smooth muscle actin. Unlike the commercially available antibody against -smooth muscle actin, GB 42 does not cross-react with -skeletal or -cardiac actins. The GB 42-antigen is expressed in smooth muscle cells, myoepithelial cells and in later stages of differentiation of myofibroblasts, in all the tissues investigated. Throughout the development of smooth muscle cells and myofibroblasts, the appearance of the GB 42-antigen occurs after the expression of vimentin, desmin and -smooth muscle actin, but prior to the expression of smooth muscle myosin. GB 42 is a reliable marker for higher stages of differentiation of smooth muscle cells and myofibroblasts.     

Placental villous stroma as a model system for myofibroblast differentiation

Different subtypes of myofibroblasts have been described according to their cytoskeletal protein patterns. It is quite likely that these different subtypes represent distinct steps of differentiation. We propose the human placental stem villi as a particularly suitable model to study this differentiation process. During the course of pregnancy, different types of placental villi develop by differentiation of the mesenchymal stroma surrounding the fetal blood vessels. In order to characterise the differentiation of placental stromal cells in the human placenta, the expression patterns of the cytoskeletal proteins vimentin, desmin, - and -smooth muscle actin, pan-actin, smooth muscle myosin, and the monoclonal antibody GB 42, a marker of myofibroblasts, were investigated on placental tissue of different gestational age (7th–40th week of gestation). Proliferation patterns were assessed with the proliferation markers MIB 1 and PCNA. Additionally, dipeptidyl peptidase IV distribution was studied in term placenta and the ultrastructure of placental stromal cells was assessed by electron microscopy. Different subpopulations of extravascular stromal cells were distinguished according to typical co-expression patterns of cytoskeletal proteins. Around the fetal stem vessels in term placental villi they were arranged as concentric layers with increasing stage of differentiation. A variable layer of extravascular stromal cells lying beneath the trophoblast expressed vimentin (V) or vimentin and desmin (VD). They were mitotically active. The next layer co-expressed vimentin, desmin, and -smooth muscle actin (VDA). More centrally towards the fetal vessels, extravascular stromal cells co-expressed vimentin, desmin, - and -smooth muscle actin, and GB 42 (VDAG). Cells close to the fetal vessels additionally co-expressed smooth muscle myosin (VDAGM). Ultrastructurally, V cells resembled typical mesenchymal cells. VD cells corresponded to fibroblasts, while VDA and VDAG cells developed features of myofibroblasts. Cells of the VDAGM-type revealed a smooth muscle cell-related ultrastructure. In earlier stages of pregnancy, stromal cell types with less complex expression patterns prevailed. The media smooth muscle cells of the fetal vessels showed a mixture of different co-expression patterns. These cells were separated from extravascular stromal cells by a layer of collagen fibres. The results obtained indicate a clearly defined spatial differentiation gradient with increasing cytoskeletal complexity in human placental stromal cells from the superficial trophoblast towards the blood vessels in the centre of the stem villi. The spatial distribution of the various stages of differentiation suggests that human placental villi could be a useful model for the study of the differentiation of myofibroblasts.