Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.     

Translation initiation and the fate of bacterial mRNAs

Studies in pro- and eukaryotes have revealed that translation can determine the stability of a given messenger RNA. In bacteria, intrinsic mRNA signals can confer efficient ribosome binding, whereas translational feedback inhibition or environmental cues can interfere with this process. Such regulatory mechanisms are often controlled by RNA-binding proteins, small noncoding RNAs and structural rearrangements within the 5' untranslated region. Here, we review molecular events occurring in the 5' untranslated region of primarily Escherichia coli mRNAs with regard to their effects on mRNA stability.     

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.     

Redifferentiation of chondrocytes and cartilage formation under intermittent hydrostatic pressure

Since articular cartilage is subjected to varying loads in vivo and undergoes cyclic hydrostatic pressure during periods of loading, it is hypothesized that mimicking these in vivo conditions can enhance synthesis of important matrix components during cultivation in vitro. Thus, the influence of intermittent loading during redifferentiation of chondrocytes in alginate beads, and during cartilage formation was investigated. A statistically significant increased synthesis of glycosaminoglycan and collagen type II during redifferentiation of chondrocytes embedded in alginate beads, as well as an increase in glycosaminoglycan content of tissue-engineered cartilage, was found compared to control without load. Immunohistological staining indicated qualitatively a high expression of collagen type II for both cases.     

Crystal structure of NblA from Anabaena sp. PCC 7120, a small protein playing a key role in phycobilisome degradation

Cyanobacterial light-harvesting complexes, the phycobilisomes, are proteolytically degraded when the organisms are starved for combined nitrogen, a process referred to as chlorosis or bleaching. Gene nblA, present in all phycobilisome-containing organisms, encodes a protein of about 7 kDa that plays a key role in phycobilisome degradation. The mode of action of NblA in this degradation process is poorly understood. Here we presented the 1.8-A crystal structure of NblA from Anabaena sp. PCC 7120. In the crystal, NblA is present as a four-helix bundle formed by dimers, the basic structural units. By using pull-down assays with immobilized NblA and peptide scanning, we showed that NblA specifically binds to the alpha-subunits of phycocyanin and phycoerythrocyanin, the main building blocks of the phycobilisome rod structure. By site-directed mutagenesis, we identified amino acid residues in NblA that are involved in phycobilisome binding. The results provided evidence that NblA is directly involved in phycobilisome degradation, and the results allowed us to present a model that gives insight into the interaction of this small protein with the phycobilisomes.     

Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.     

Similarities,Differences and Synergisms Between HERA and LCA—An Analysis at Three Levels

Linkages between Human and Environmental Risk Assessment (HERA) and Life-Cycle Assessment (LCA) can be analyzed at three levels: the basic equations to describe environmental behavior and dose-response relationships of chemicals; the overall model structure of these tools; and the applications of the tools. At level 1 few differences exist: both tools use essentially the same fate and effect models, including their coefficients and data. At level 2 distinctive differences emerge: regional or life-cycle perspective, emission pulses or fluxes, scope of chemicals and types of impacts, use of characterization factors, spatial and temporal detail, aggregation of effects, and the functional unit as basis of the assessment. Although the two tools typically differ in all these aspects, only the functional unit issue renders the tools fundamentally different, expressing itself also in some main characteristics of the modeling structure. This impedes full integration, which is underpinned in mathematical terms. At level 3 the aims of the tools are complementary: quantified risk estimates of chemicals for HERA versus quantified product assessment for LCA. Here, beneficial synergism is possible between the two tools, as illustrated by some cases. These also illustrate that where full integration is suggested, in practice this is not achieved, thus in fact supporting the conclusions.     

Renal IL-17 expression in human ANCA-associated glomerulonephritis

Interleukin-17A (IL-17) promotes inflammatory renal tissue damage in mouse models of crescentic glomerulonephritis, including murine experimental autoimmune anti-myeloperoxidase glomerulonephritis, which most likely depends on IL-17-producing Th17 cells. In human anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, however, the cellular sources of IL-17 remain to be elucidated. Therefore, we analyzed human kidney biopsies of active necrotizing and crescentic ANCA-associated glomerulonephritis by immunohistochemistry using an IL-17-specific antibody and by immunofluorescent colocalization with cell type markers. We detected numerous IL-17-expressing (IL-17(+)) cells in the glomeruli and in the tubulointerstitium. Unexpectedly, most of these IL-17(+) cells were polymorphonuclear neutrophilic granulocytes, while IL-17(+) T cells and IL-17(+) mast cells were present at significantly lower frequencies. IL-17 was not detected in other infiltrating or resident kidney cells. In those patients who had not received immunosuppressive treatment before biopsy, serum creatinine levels were positively correlated with tubulointerstitial IL-17(+) neutrophils as well as IL-17(+) T cells. Furthermore, we could demonstrate that purified human blood neutrophils expressed IL-17 protein and released it upon stimulation in vitro. In conclusion, these results support a pathogenic role for IL-17 in human ANCA-associated glomerulonephritis. Our data suggest that in the acute stage of the disease neutrophils may act as an important immediate-early innate source of IL-17 and may thereby initiate and promote ongoing renal inflammation. IL-17 may thus be a target for treating acute ANCA-associated glomerulonephritis.