P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.     

Anatomical heterogeneity of memory CD4+ T cells due to reversible adaptation to the microenvironment

The memory T cell pool is characterized by a substantial degree of heterogeneity in phenotype and function as well as anatomical distribution, but the underlying mechanisms remain unclear. In this study we confirm that the memory CD4(+) T cell pool in wild-type and TCR-transgenic mice consists of heterogeneous subsets, as defined by surface marker expression or cytokine production. Extralymphoid sites contain significant numbers of memory CD4(+) T cells, which are phenotypically and functionally distinct from their lymphoid counterparts. However, we show in this study that the phenotype of lymphoid and extralymphoid memory T cells is not stable. Instead, the unique properties of extralymphoid memory T cells are acquired upon migration into extralymphoid sites and are lost when memory T cells migrate back into lymphoid organs. Thus, at least some of the extralymphoid properties may represent a transient activation state that can be adopted by T cells belonging to a single memory T cell pool. Furthermore, such intermittent activation during or after migration into extralymphoid sites could provide an important signal, promoting the survival and functional competence of memory T cells in the absence of Ag.     

Adducin Is Involved in Endothelial Barrier Stabilization

Adducins tightly regulate actin dynamics which is critical for endothelial barrier function. Adducins were reported to regulate epithelial junctional remodeling by controlling the assembly of actin filaments at areas of cell-cell contact. Here, we investigated the role of α-adducin for endothelial barrier regulation by using microvascular human dermal and myocardial murine endothelial cells. Parallel transendothelial electrical resistance (TER) measurements and immunofluorescence analysis revealed that siRNA-mediated adducin depletion impaired endothelial barrier formation and led to severe fragmentation of VE-cadherin immunostaining at cell-cell borders. To further test whether the peripheral localization of α-adducin is functionally linked with the integrity of endothelial adherens junctions, junctional remodeling was induced by a Ca²⁺-switch assay. Ca²⁺-depletion disturbed both linear vascular endothelial (VE)-cadherin and adducin location along cell junctions, whereas their localization was restored following Ca²⁺-repletion. Similar results were obtained for α-adducin phosphorylated at a site typical for PKA (pSer481). To verify that endothelial barrier properties and junction reorganization can be effectively modulated by altering Ca²⁺-concentration, TER measurements were performed. Thus, Ca²⁺-depletion drastically reduced TER, whereas Ca²⁺-repletion led to recovery of endothelial barrier properties resulting in increased TER. Interestingly, the Ca²⁺-dependent increase in TER was also significantly reduced after efficient α-adducin downregulation. Finally, we report that inflammatory mediator-induced endothelial barrier breakdown is associated with loss of α-adducin from the cell membrane. Taken together, our results indicate that α-adducin is involved in remodeling of endothelial adhesion junctions and thereby contributes to endothelial barrier regulation.     

Colonization of experimentally created gaps along an alpine successional gradient

The colonization of artificially created gaps was analyzed along an alpine successional gradient from pioneer to early, late, and old successional stages. The presence/absence of species and the abundances of seedlings and adults in the gaps were recorded and compared with those of the surrounding areas. We hypothesized that in the older successional stages, the gaps were likely to be colonized by clonal ingrowth of the surrounding species. In the younger stages, we expected to find a high presence of seedlings and adults recruited by seeds. Micro-succession in the gaps occurred at each successional stage, with all life forms among the colonizers. The abundance of seedlings was significantly higher in the gaps compared with the surrounding area. At the early and late successional stages, the surrounding areas provided safe sites for seedling establishment, with the abundance of adults recruited by seeds higher at the gap edges than in the gap centers. We can confirm the first hypothesis of a higher clonal ingrowth in the old successional stage. Clonal ingrowth also occurred in the younger successional stages. Despite the lower species richness in the gaps, a positive correlation was found between gap and surrounding species frequencies, which were the highest in the pioneer and the lowest in the old successional stage. We conclude that gaps are relevant for seedling recruitment along the entire primary succession gradient. New species invasions from greater distances were not observed in the gaps. The dominant species on each site were identified to be successful gap colonizers.     

Nucleotide docking: prediction of reactant state complexes for ribonuclease enzymes

Ribonuclease enzymes (RNases) play key roles in the maturation and metabolism of all RNA molecules. Computational simulations of the processes involved can help to elucidate the underlying enzymatic mechanism and is often employed in a synergistic approach together with biochemical experiments. Theoretical calculations require atomistic details regarding the starting geometries of the molecules involved, which, in the absence of crystallographic data, can only be achieved from computational docking studies. Fortunately, docking algorithms have improved tremendously in recent years, so that reliable structures of enzyme–ligand complexes can now be successfully obtained from computation. However, most docking programs are not particularly optimized for nucleotide docking. In order to assist our studies on the cleavage of RNA by the two most important ribonuclease enzymes, RNase A and RNase H, we evaluated four docking tools—MOE2009, Glide 5.5, QXP-Flo+0802, and Autodock 4.0—for their ability to simulate complexes between these enzymes and RNA oligomers. To validate our results, we analyzed the docking results with respect to the known key interactions between the protein and the nucleotide. In addition, we compared the predicted complexes with X-ray structures of the mutated enzyme as well as with structures obtained from previous calculations. In this manner, we were able to prepare the desired reaction state complex so that it could be used as the starting structure for further DFT/B3LYP QM/MM reaction mechanism studies.     

Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion

Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in?a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in?vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool.     

Hybrid rye performance under natural drought stress in Europe

Several rye growing regions of Central Europe suffered from severe drought stress in the last decade. Rye is typically grown on sandy soils with low water-holding capacity in areas with low rainfall, thus drought-tolerant varieties are urgently needed. The main objective of our study was to evaluate the drought stress tolerance of rye hybrids using large-scaled field experiments. Two biparental populations (Pop-A, Pop-B) each consisting of 220 F_2:4 lines from the Petkus gene pool and their parents were evaluated for grain yield testcross performance under irrigated (I) and rainfed (R) regime in six environments. We observed for most environments severe drought stress leading to an average grain yield reduction of 23.8 % for rainfed compared to irrigated regime in drought stress environments. A decomposition of the variance revealed significant (P < 0.01) genotypic and genotype × environment interaction variances but only a minor effect of drought stress on the ranking of the genotypes with regard to grain yield. In conclusion, separate breeding programs for drought-tolerant genotypes are not superior to the currently practiced selection under rainfed conditions without irrigation in hybrid rye breeding in Central Europe.     

Phenotypic and Functional Profiling of CD4 T Cell Compartment in Distinct Populations of Healthy Adults with Different Antigenic Exposure

Background

Multiparameter flow cytometry has revealed extensive phenotypic and functional heterogeneity of CD4 T cell responses in mice and humans, emphasizing the importance of assessing multiple aspects of the immune response in correlation with infection or vaccination outcome. The aim of this study was to establish and validate reliable and feasible flow cytometry assays, which will allow us to characterize CD4 T cell population in humans in field studies more fully.

Methodology/Principal Findings

We developed polychromatic flow cytometry antibody panels for immunophenotyping the major CD4 T cell subsets as well as broadly characterizing the functional profiles of the CD4 T cells in peripheral blood. We then validated these assays by conducting a pilot study comparing CD4 T cell responses in distinct populations of healthy adults living in either rural or urban Kenya. This study revealed that the expression profile of CD4 T cell activation and memory markers differed significantly between African and European donors but was similar amongst African individuals from either rural or urban areas. Adults from rural Kenya had, however, higher frequencies and greater polyfunctionality among cytokine producing CD4 T cells compared to both urban populations, particularly for “Th1” type of response. Finally, endemic exposure to malaria in rural Kenya may have influenced the expansion of few discrete CD4 T cell populations with specific functional signatures.

Conclusion/Significance

These findings suggest that environmentally driven T cell activation does not drive the dysfunction of CD4 T cells but is rather associated with greater magnitude and quality of CD4 T cell response, indicating that the level or type of microbial exposure and antigenic experience may influence and shape the functionality of CD4 T cell compartment. Our data confirm that it is possible and mandatory to assess multiple functional attributes of CD4 T cell response in the context of infection.     

Sex and dosing-time dependencies in irinotecan-induced circadian disruption

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50 mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2?h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers.     

Glomerulometric data in experimental toxicological investigations

Glomerulometric quantitative investigations from rat kidney during chronic attempt with the substances Deta-20 and Polystabil VZ on paraffin sections were carried out. These substances are utilized in textile and food industry. Data, obtained by means of a graduated eyepiece-micrometer for light microscope, were plotted on histograms. This work is an attempt to introduce lightoptical morphometric measurements for nuclei and cells as well as for other structures (glomeruli).