Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells

Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin.     

RNAi Transfection Results in Lipidome Changes

RNAi experiments are ubiquitously used in cell biology and are achieved by transfection of small interfering RNAs (siRNAs) into cells using a transfection reagent. These results in knock‐down of proteins of interest, and the phenotypic consequences are then analyzed. It is reported here that two common RNA interference (RNAi) transfection reagents, DharmaFECT 1 and INTERFERin, in mock transfections using non‐targeting siRNAs, cause alterations in the lipidome of HeLa cells. Some lipids change in response to both, presumably chemically different, transfection reagents, while other lipid species change only in response to one of the reagents. While the functional implications of these lipidomic alterations remain to be investigated, the authors' experiments suggest that it is important to use appropriate mock transfection controls during RNAi experiments, ideally complemented by an orthogonal perturbation, especially when investigating membrane‐associated phenomena.     

Two novel human and mouse DNA polymerases of the polX family

We describe here two novel mouse and human DNA polymerases: one (pol λ) has homology with DNA polymerase β while the other one (pol µ) is closer to terminal deoxynucleotidyltransferase. However both have DNA polymerase activity in vitro and share similar structural organization, including a BRCT domain, helix–loop–helix DNA-binding motifs and polymerase X domain. mRNA expression of pol λ is highest in testis and fetal liver, while expression of pol µ is more lymphoid, with highest expression both in thymus and tonsillar B cells. An unusually large number of splice variants is observed for the pol µ gene, most of which affect the polymerase domain. Expression of mRNA of both polymerases is down-regulated upon treatment by DNA damaging agents (UV light, γ-rays or H₂O₂). This suggests that their biological function may differ from DNA translesion synthesis, for which several DNA polymerase activities have been recently described. Possible functions are discussed.     

Variable Cell Morphology Approach for Individual-Based Modeling of Microbial Communities

An individual-based, mass-spring modeling framework has been developed to investigate the effect of cell properties on the structure of biofilms and microbial aggregates through Lagrangian modeling. Key features that distinguish this model are variable cell morphology described by a collection of particles connected by springs and a mechanical representation of deformable intracellular, intercellular, and cell-substratum links. A first case study describes the colony formation of a rod-shaped species on a planar substratum. This case shows the importance of mechanical interactions in a community of growing and dividing rod-shaped cells (i.e., bacilli). Cell-substratum links promote formation of mounds as opposed to single-layer biofilms, whereas filial links affect the roundness of the biofilm. A second case study describes the formation of flocs and development of external filaments in a mixed-culture activated sludge community. It is shown by modeling that distinct cell-cell links, microbial morphology, and growth kinetics can lead to excessive filamentous proliferation and interfloc bridging, possible causes for detrimental sludge bulking. This methodology has been extended to more advanced microbial morphologies such as filament branching and proves to be a very powerful tool in determining how fundamental controlling mechanisms determine diverse microbial colony architectures.     

Ways of Asking,Ways of Telling

The interpretive understanding that can be derived from interviews is highly influenced by methods of data collection, be they structured or semistructured, ethnographic, clinical, life-history or survey interviews. This article responds to calls for research into the interview process by analyzing data produced by two distinctly different types of interview, a semistructured ethnographic interview and the Structured Clinical Interview for DSM, conducted with participants in the Navajo Healing Project. We examine how the two interview genres shape the context of researcher-respondent interaction and, in turn, influence how patients articulate their lives and their experience in terms of illness, causality, social environment, temporality and self/identity. We discuss the manner in which the two interviews impose narrative constraints on interviewers and respondents, with significant implications for understanding the jointly constructed nature of the interview process. The argument demonstrates both divergence and complementarity in the construction of knowledge by means of these interviewing methods.     

Normal immune system development in mice lacking the Deltex-1 RING finger domain

The Notch signaling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1(Delta/Delta) mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular those from a fourth Deltex gene identified during the course of this study, are also discussed.     

Engineering of vascularized adipose constructs

Adipose tissue engineering offers a promising alternative to the current surgical techniques for the treatment of soft tissue defects. It is a challenge to find the appropriate scaffold that not only represents a suitable environment for cells but also allows fabrication of customized tissue constructs, particularly in breast surgery. We investigated two different scaffolds for their potential use in adipose tissue regeneration. Sponge-like polyurethane scaffolds were prepared by mold casting with methylal as foaming agent, whereas polycaprolactone scaffolds with highly regular stacked-fiber architecture were fabricated with fused deposition modeling. Both scaffold types were seeded with human adipose tissue-derived precursor cells, cultured and implanted in nude mice using a femoral arteriovenous flow-through vessel loop for angiogenesis. In vitro, cells attached to both scaffolds and differentiated into adipocytes. In vivo, angiogenesis and adipose tissue formation were observed throughout both constructs after 2 and 4?weeks, with angiogenesis being comparable in seeded and unseeded constructs. Fibrous tissue formation and adipogenesis were more pronounced on polyurethane foam scaffolds than on polycaprolactone prototyped scaffolds. In conclusion, both scaffold designs can be effectively used for adipose tissue engineering.     

Referate

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