Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.     

pH-optima in lipase-catalysed esterification

Though lipases are frequently applied in ester synthesis, fundamental information on optimal pH or substrate concentration, can almost only be found for the reverse reaction - hydrolysis. This study demonstrates that the pH-optima of lipase-catalysed esterifications differ significantly from the optima of the hydrolysis reaction. In the esterification of n-butanol and propionic acid with lipases of Candida rugosa (CRL) and Thermomyces lanuginosa (TLL) pH-optima of 3.5 and 4.25, respectively, were found. This is about 3-4 units (CRL) and 7 units (TLL) in pH lower than optimum for hydrolysis. Enzyme activity increased with increasing concentrations of protonated acid indicating that the protonated acid rather than the deprotonated form is substrate for esterification. The rate of esterification can be drastically increased by ensuring acid concentrations up to 1000 mmol L^-1 for CRL and 600 mmol L^-1 for TLL in the reaction system.     

Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system

The identification of mesenchymal stem cells (MSCs) in adult human tissues and the disclosure of their self-renew-al and multi-lineage differentiation capabilities have provided exciting prospects for cell-based regeneration and tis-sue engineering. Although a considerable amount of data is available describing MSCs, there is still lack of information regarding the molecular mechanisms that govern their adhesion and migration. In this work, we will review the current state of knowledge on integrins and other adhesion molecules found to be expressed on MSCs. The discussed topics include the characteristics of MSCs and their clinical applications, integrins and their central role in cell-matrix attachment and migration, and comments on mobilization, differentiation and contribution to tumour development. Finally, by understanding the complex and fundamental pathways by which MSCs attach and migrate, it might be possible to fine-tune the strategies for effective and safe use of MSCs in regenerative therapies.     

Identification and Application of Plasmids Suitable for Transfer of Foreign DNA to Members of the Genus Gordonia

Gene transfer systems for Gordonia polyisoprenivorans strains VH2 and Y2K based on electroporation and conjugation, respectively, were established. Several parameters were optimized, resulting in transformation efficiencies of >4 × 10⁵ CFU/μg of plasmid DNA. In contrast to most previously described electroporation protocols, the highest efficiencies were obtained by applying a heat shock after the intrinsic electroporation. Under these conditions, transfer and autonomous replication of plasmid pNC9503 was also demonstrated to proceed in G. alkanivorans DSM44187, G. nitida DSM44499^T, G. rubropertincta DSM43197^T, G. rubropertincta DSM46038, and G. terrae DSM43249^T. Conjugational plasmid DNA transfer to G. polyisoprenivorans resulted in transfer frequencies of up to 5 × 10⁻⁶ of the recipient cells. Recombinant strains capable of polyhydroxyalkanoate synthesis from alkanes were constructed.     

Force-dependent stepping kinetics of myosin-V

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at approximately 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.     

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.     

Ring folding in cyclopentadienyl diazabutadiene complexes of group 4 and 5 transition metals

The syntheses of CpM(i-PrDAB)₂ (M = Nb, Ta; Cp = (C₅H₅); i-PrDAB = bis-isopropyl-1,4-diazabuta-1,3-diene) are reported. Both show fluxional NMR spectra indicating that the two DAB rings differ. The X-ray crystal structure of CpNb(i-PrDAB)₂ shows one ring to be more folded than the other. Density functional calculations have been used to investigate the degree of folding of the chelate ring in the compounds Cp₂M(R-DAB), (M = Ti, Zr, Hf, Nb, Ta; R = H, i-Pr; DAB = 1,4-diazabuta-1,3-diene) and CpM(R-DAB)₂ (M = Nb and Ta). For Cp₂M(R-DAB) the group 4 compounds all have folded rings whereas the Nb and Ta compounds have planar rings. In all compounds the rings are reduced and the folding is driven by the electron number requirements of the metal centre.