Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro.

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.     

α/β-Hydrolase Domain Containing Protein 15 (ABHD15) – an Adipogenic Protein Protecting from Apoptosis

Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.     

Preoperative Diffusion-Weighted Imaging of Single Brain Metastases Correlates with Patient Survival Times

Background

MRI-based diffusion-weighted imaging (DWI) visualizes the local differences in water diffusion in vivo. The prognostic value of DWI signal intensities on the source images and apparent diffusion coefficient (ADC) maps respectively has not yet been studied in brain metastases (BM).

Methods

We included into this retrospective analysis all patients operated for single BM at our institution between 2002 and 2010, in whom presurgical DWI and BM tissue samples were available. We recorded relevant clinical data, assessed DWI signal intensity and apparent diffusion coefficient (ADC) values and performed histopathological analysis of BM tissues. Statistical analyses including uni- and multivariate survival analyses were performed.

Results

65 patients (34 female, 31 male) with a median overall survival time (OS) of 15 months (range 0–99 months) were available for this study. 19 (29.2%) patients presented with hyper-, 3 (4.6%) with iso-, and 43 (66.2%) with hypointense DWI. ADCmean values could be determined in 32 (49.2%) patients, ranged from 456.4 to 1691.8*10⁻⁶ mm²/s (median 969.5) and showed a highly significant correlation with DWI signal intensity. DWI hyperintensity correlated significantly with high amount of interstitial reticulin deposition. In univariate analysis, patients with hyperintense DWI (5 months) and low ADCmean values (7 months) had significantly worse OS than patients with iso/hypointense DWI (16 months) and high ADCmean values (30 months), respectively. In multivariate survival analysis, high ADCmean values retained independent statistical significance.

Conclusions

Preoperative DWI findings strongly and independently correlate with OS in patients operated for single BM and are related to interstitial fibrosis. Inclusion of DWI parameters into established risk stratification scores for BM patients should be considered.     

Java editor for biological pathways

SUMMARY: A visual Java-based tool for drawing and annotating biological pathways was developed. This tool integrates the possibilities of charting elements with different attributes (size, color, labels), drawing connections between elements in distinct characteristics (color, structure, width, arrows), as well as adding links to molecular biology databases, promoter sequences, information on the function of the genes or gene products, and references. It is easy to use and system independent. The result of the editing process is a PNG (portable network graphics) file for the images and XML (extended markup language) file for the appropriate links.     

Distribution and origin of steroid hormones in the yolk of Japanese quail eggs (Coturnix coturnix japonica)

The yolk of avian eggs contains steroid hormones, which may influence the development and behaviour of hatched birds. The aim of the present study was to investigate the concentration as well as the distribution of various gonadal steroids in the yolk spheres of quail eggs. Steroid concentrations of dissected yolk layers were analysed after alcoholic extraction using enzyme immunoassays (EIAs) for progesterone, androstenedione and testosterone. To monitor the uptake of testosterone into the yolk, radioactive testosterone was injected i.m. into six female quails. The radioactivity of yolk layers of subsequently laid eggs was measured by liquid scintillation counting. Progesterone concentrations were highest in the outer layer (median: 2265 nmol/kg). Androstenedione (median: 453 nmol/kg), as the major androgen, and testosterone (median: 99 nmol/kg) reached their highest concentrations in interior layers, whereas in the centre the concentration of all three hormones was low. No significant variation of steroid levels in yolk layers of subsequently laid eggs was found. The highest radioactivity was detected in the outer yolk layer in those eggs laid 1 day after injection and in subsequently laid eggs was measured nearer to the centre. These results indicated local origin of the steroid hormones especially because of the result that only 0.1% of the radioactivity entered the yolk. We conclude that steroid concentrations in the yolk layers reflected progesterone and androgen production of the cells of the follicular wall at the time.     

Inhibition of DNA-dependent protein kinase induces accelerated senescence in irradiated human cancer cells

DNA-dependent protein kinase (DNA-PK) plays a pivotal role in the repair of DNA double-strand breaks (DSB) and is centrally involved in regulating cellular radiosensitivity. Here, we identify DNA-PK as a key therapeutic target for augmenting accelerated senescence in irradiated human cancer cells. We find that BEZ235, a novel inhibitor of DNA-PK and phosphoinositide 3-kinase (PI3K)/mTOR, abrogates radiation-induced DSB repair resulting in cellular radiosensitization and growth delay of irradiated tumor xenografts. Importantly, radiation enhancement by BEZ235 coincides with a prominent p53-dependent accelerated senescence phenotype characterized by positive β-galactosidase staining, G(2)-M cell-cycle arrest, enlarged and flattened cellular morphology, and increased p21 expression and senescence-associated cytokine secretion. Because this senescence response to BEZ235 is accompanied by unrepaired DNA DSBs, we examined whether selective targeting of DNA-PK also induces accelerated senescence in irradiated cells. Significantly, we show that specific pharmacologic inhibition of DNA-PK, but not PI3K or mTORC1, delays DSB repair leading to accelerated senescence after radiation. We additionally show that PRKDC knockdown using siRNA promotes a striking accelerated senescence phenotype in irradiated cells comparable with that of BEZ235. Thus, in the context of radiation treatment, our data indicate that inhibition of DNA-PK is sufficient for the induction of accelerated senescence. These results validate DNA-PK as an important therapeutic target in irradiated cancer cells and establish accelerated senescence as a novel mechanism of radiosensitization induced by DNA-PK blockade.     

Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation

How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor–recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.     

The effect of temperature on DNA structural transitions under the action of Cu2+ and Ca2+ ions in aqueous solutions

The work examines the structural transitions of DNA under the action of Cu2+ and Ca2+ ions in aqueous solution at temperatures of 29 and 45 degrees C by ir spectroscopy. Upon binding to the divalent ions studied, DNA transits into the compact state both at 29 and 45 degrees C. In the compact state DNA remains in B-form limits. The compaction process is of high positive cooperativity. As temperature increases the divalent metal ion concentration required to induce DNA compaction decreases in the case of Cu(2+)-induced compaction and increases in the case of Ca(2+)-induced compaction. It is suggested that the mechanism of the temperature effect on DNA compaction in the presence of Cu2+ ions possessing higher affinity for DNA bases differs from that of the temperature influence on Ca(2+)-induced DNA compaction. In the case of copper ions the determining factor is the increase of binding constants of the Cu2+ ions interacting with the denatured parts formed on DNA while in the case of calcium ions it is the decreased screening action of counterions upon the increase of their hydration with temperature. The efficiency of divalent metal ions studied in inducing DNA compaction depends on hydration of counterions. DNA compaction occurs in a narrow interval of Cu2+ concentrations. As the Cu2+ ion concentration increases, DNA compaction is replaced with Cu(2+)-induced DNA aggregation. At elevated temperatures Cu(2+)-induced DNA compaction could acquire a phase transition character.     

Diet selection by hares (Lepus europaeus) in arable land and its implications for habitat management

Populations of European hares (Lepus europaeus) have experienced a dramatic decline throughout Europe in recent decades. European hares are assumed to prefer weeds over arable crops, and weed abundance was reduced by the intensification of agriculture. Therefore, modern agriculture has been blamed as a major factor affecting European hare populations. However, it is questionable whether European hares select weeds at all, as previous studies had major methodological limitations. By comparing availability and use of plants with Chesson’s Electivity Index, we investigated whether the European hare actually feeds selectively on different plants in arable land. Food availability and use were dominated by cultivated crops (e.g. winter wheat, spring barley and sugar beet). Diet selection analysis revealed that in autumn and winter, European hares predominantly preferred cultivated crops (winter wheat) and food items provided by hunters (tubers of sugar beet and carrot). In spring and summer, apart from soy, only weeds (e.g. clover and corn poppy) were positively selected, especially after cereal crops were harvested. We suggest that the decline in European hare populations throughout Europe was facilitated by the decrease in weed abundance. Wildlife-friendly set-asides in arable land have the potential to reconcile the European Union’s Common Agricultural Policy with wildlife conservation.