A novel Cripto-related protein reveals an essential role for EGF-CFCs in Nodal signalling in Xenopus embryos

The location, timing and intensity of Nodal signalling are all critical for proper patterning of the vertebrate embryo. Genetic evidence from mouse and zebrafish indicates that EGF-CFC family members are essential for Nodal ligands to signal. However, the Xenopus EGF-CFC, FRL1, has been implicated in Wnt signalling and in activation of Erk MAP kinase. Here, we identify two additional Xenopus EGF-CFCs, XCR2 and XCR3. We have focused on the role of XCR1/FRL1 and XCR3, which are both expressed at gastrula stages when Nodal signalling is active. We demonstrate spatial and temporal regulation of XCR1 protein expression, whereas XCR3 appears to be expressed ubiquitously. Using gain and loss of function approaches, we show that XCR1 and XCR3 are required for Nodal-related ligands to signal during early Xenopus development. Moreover, different Nodal-related ligands require different XCRs to signal. When both XCR1 and XCR3 are knocked down, activation of the Nodal intracellular signal transducer, Smad2, is severely inhibited and neither gastrulation nor mesendoderm formation occurs. Together our results indicate that the XCRs are important for modulation of the timing and intensity of Nodal signalling in Xenopus embryos.     

Different modulation of TRAIL-induced apoptosis by inhibition of pro-survival pathways in TRAIL-sensitive and TRAIL-resistant colon cancer cells

Epithelial cells can be manipulated to undergo apoptosis depending on the balance between pro-survival and apoptotic signals. We showed that TRAIL-induced apoptosis may be differentially regulated by inhibitors of MEK ERK (U0126) or PI3K/Akt (LY294002) pathway in TRAIL-sensitive (HT-29) and TRAIL-resistant (SW620) human epithelial colon cancer cells. U0126 or LY294002 significantly enhanced TRAIL-induced apoptosis in HT-29 cells, but not in SW620 cells. We report a different regulation of the level of an anti-apoptotic Mcl-1 protein under MEK/ERK or PI3K/Akt pathway inhibition and suggest the mechanisms involved. A special attention was paid to the role of the ERK1/2, Akt, and glycogen synthase kinase 3beta.     

The effect of a sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice

To unravel the molecular mechanisms mediating the effects of androgens on spermatogenesis, testicular gene expression was compared in mice with Sertoli cell-selective androgen receptor knockout (SCARKO) and littermate controls on postnatal d 10. Microarray analysis identified 692 genes with significant differences in expression. Of these, 28 appeared to be down-regulated and 12 up-regulated at least 2-fold in SCARKOs compared with controls. For nine of the more than 2-fold down-regulated genes, androgen regulation was confirmed by treatment of wild-type mice with an antiandrogen (flutamide). Some of them were previously described to be androgen regulated or essential for spermatogenesis. Serine-type protease inhibitors were markedly overrepresented in this down-regulated subgroup. A time study (d 8-20), followed by cluster analysis, allowed identification of distinct expression patterns of differentially expressed genes. Three genes with a pattern closely resembling that of Pem, a prototypical androgen-regulated gene expressed in Sertoli cells, were selected for confirmation by quantitative RT-PCR and additional analysis. The data confirm that the SCARKO model allows identification of novel androgen-regulated genes in the testis. Moreover, they suggest that protease inhibitors and other proteins related to tubular restructuring and cell junction dynamics may be controlled in part by androgens.     

Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex

We imaged axons in layer (L) 1 of the mouse barrel cortex in vivo. Axons from thalamus and L2/3/5, or L6 pyramidal cells were identified based on their distinct morphologies. Their branching patterns and sizes were stable over times of months. However, axonal branches and boutons displayed cell type-specific rearrangements. Structural plasticity in thalamocortical afferents was mostly due to elongation and retraction of branches (range, 1-150 microm over 4 days; approximately 5% of total axonal length), while the majority of boutons persisted for up to 9 months (persistence over 1 month approximately 85%). In contrast, L6 axon terminaux boutons were highly plastic (persistence over 1 month approximately 40 %), and other intracortical axon boutons showed intermediate levels of plasticity. Retrospective electron microscopy revealed that new boutons make synapses. Our data suggest that structural plasticity of axonal branches and boutons contributes to the remodeling of specific functional circuits.     

Kinetics of total enzymatic hydrolysis of acetylcholine and acetylthiocholine

Kinetics and the mechanism of total in vitro hydrolyses (i.e. up to the exhaustion of substrate) of acetylcholine and acetylthiocholine by acetylcholinesterase and butyrylcholinesterase were studied in vitro in a batch reactor at 25 degrees C, pH 8 and ionic strength of 0.11 M. Every hydrolysis was monitored by 2-3 independent analytical methods. All studied types of enzymatic hydrolyses fulfilled the Michaelis-Menten reaction scheme with the irreversible second step. A table of obtained average values of rate constants and estimations of initial molar enzyme concentrations, and discussion of the results are presented.     

To the intranucleolar translocation of AgNORs in leukemic early granulocytic and plasmacytic precursors

Early leukemic granulocytic and plasmacytic precursors were studied in vitro and in vivo to provide an information on the intranucleolar distribution of AgNORs (silver stained nucleolus organizer regions). In most of these cells AgNORs appeared as clusters of silver stained particles distributed in the whole nucleolar body. On the other hand, in some leukemic early granulocytic precursors, i.e., in myeloblasts and promyelocytes enlarged AgNORs were translocated in the nucleolar peripheral part. In addition, the number of translocated AgNORs at the nucleolar periphery was significantly smaller. Such translocation of a reduced number of AgNORs was easily produced by experimental aging, i.e., starving of cultured leukemic early granulocytic precursors (HL-60 and K562 cells) in vitro and seems to be reversible. Similar translocation of a reduced number of AgNORs was also produced by aging of leukemic plasmacytic precursors. Thus, the translocation of the reduced number of AgNORs to the nucleolar periphery in some blastic leukemic hematopoietic cells might be an useful marker of their aging at the single cell level. However, more studies in this direction are required in the future.     

Nutrition and theory of mind--The role of polyunsaturated fatty acids (PUFA) in the development of theory of mind

Breast-milk provides nutrients required for the development of the brain. n-6 and n-3 long-chain polyunsaturated fatty acids (LCPUFAs) have been suggested to be particularly involved. In this study levels of fatty acids in breast-milk were examined in relation to theory of mind (ToM) (n = 13) and WISC-III (n = 22) in six-year-old children. ToM tasks comprised four illustrated stories with questions about emotional (sad) events. Single polyunsaturated fatty acids (PUFA) were estimated as well as ratios between different fatty acids in order to describe putative associations between PUFA and psychological measures. Results show correlations between both ToM and WISC-III with single n-6 PUFA and the ratios DHA/AA and DHA/DPA. The correlations remained when socio-demographic factors were statistically controlled for. The positive findings related to the n-6 and n-3 LCPUFAs corroborate previous findings related to child cognitive development.     

Transfection with steroid-responsive reporter constructs shows glucocorticoid rather than androgen responsiveness in cultured Sertoli cells

It remains unclear why it has proven so difficult to identify androgen target genes in cultured Sertoli cells. Given the lack of useful endogenous reporter genes, we studied the androgen and glucocorticoid responsiveness of these cells by transfection with three different steroid-responsive reporter constructs. The constructs were driven by the tyrosine aminotransferase steroid-responsive region (TAT-GRE4x-Luc), the mouse mammary tumor virus promoter (MMTV-Luc) and the Pem homeobox gene proximal promoter respectively (Pem-Luc). These constructs can be activated either by both the glucocorticoid receptor (GR) and the androgen receptor (AR) (TAT-GRE4x-Luc and MMTV-Luc) or selectively by the AR (Pem-Luc). Despite high transfection efficiency (30-40%) none of the constructs could be activated by treatment of the Sertoli cells with testosterone, 5alpha-dihydrotestosterone or synthetic androgens. Even pretreatment with follicle-stimulating hormone to raise AR levels (from 31 up to 82fmol/mg protein) did not result in androgen responsiveness. In contrast, treatment with dexamethasone markedly stimulated TAT-GRE4x-Luc and MMTV-Luc activity. GR levels reached a value of 172fmol/mg protein in the cultured cells and both AR and GR displayed homogeneous distribution by immunocytochemical evaluation. Androgen responsiveness was restored and glucocorticoid responsiveness was increased by cotransfection with AR or GR expression constructs. Under cotransfection conditions, 1nM of testosterone (a concentration that is some 100 times lower than that estimated to be present in the testis) was sufficient to stimulate the TAT-GRE4x-Luc maximally. Our data indicate that cultured Sertoli cells respond better to glucocorticoids than to androgens and that one of the factors limiting androgen responsiveness is the availability of AR. Other factors limiting the transactivation capacity of the (endogenous) AR, however, cannot be excluded.     

Restored meadow harvestman communities (Opiliones) in the Bílé Karpaty Protected Landscape Area, Czech Republic

Harvestman communities inhabiting plots treated differently for grassland restoration were investigated at the Vyzkum site near Malá Vrbka village (Bílé Karpaty Protected Landscape Area). Harvestman were sampled by pitfall trapping from 1999 to 2003 on plots sown with native haymeadow seed mixture, also on plots where narrow strips of regional seed mixtures were sown either within a matrix of commercial grass mixture or within vegetation cover in natural regeneration state and on plots in a natural regeneration state. Additionally, harvestman were collected in a field under permanent crop rotation and in a neighbouring xerothermic deciduous forest. In total, 5,086 individuals of harvestman representing 15 species from three families were obtained. Phalangium opilio was dominant (78%) and P. opilio, Rilaena triangularis and Zachaeus crista were the most frequent species. The results confirmed colonisation and subsequent development of harvestman communities on meadows in various state of restoration, including plots with spontaneous plant succession. Nevertheless, biotope character and successive formation of plant cover evidently influenced the structure of harvestman communities. The highest number of taxa (12) was recorded on plots with natural regeneration; the lowest one (9) was recorded in the field with permanent crop rotation. The highest values of diversity and equitability indices of harvestman communities were found in neighbouring forest habitats representing possible sources of harvestman migration.     

Biosynthesis and metabolic pathways of pivalic acid

Occurrence, biosynthesis, and biodegradation of pivalic acid and other compounds, having a quaternary carbon atom by different bacteria, are described. We have summarized the relevant data that have so far been published, presenting them in a graphical form, i.e., as biodegradation pathways including B(12)-dependent isomerization and desaturation that lead to the degradation of pivalic acid and similar compounds to products with other than quaternary carbon atoms, i.e., compounds whose catabolism is well known.