Proliferation status defines functional properties of endothelial cells

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell–cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi’s sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.     

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.     

Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10

A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10-TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.     

Temperature and high salinity effects in germinating dimorphic seeds of Atriplex rosea

Atriplex rosea L. (Chenopodiaceae; tumbling orach), an annual herb, is a widely established weedy species of disturbed sites in all counties of Utah. Seeds of Atriplex rosea were collected from a salt marsh in Faust, Utah, and are dimorphic, light brown, and 2-2.5 mm wide, or black and 1-2 mm wide. Seed germination responses of the black and brown seeds were studied over a range of salinity and temperature. Both brown and black seeds germinated at 1000 mM NaCl, and the optimal temperature for germination of both types was 20°-30°C. Variation in temperature, however, affected germination of black seeds more than brown seeds. At lower thermoperiod only 40%-50% black seeds germinated in nonsaline control, and germination was almost completely inhibited with the inclusion of salinity. However, all brown seeds germinated in control at temperatures above 5°-15°C, and inhibition caused by salinity was comparatively lower. Brown seeds had a higher germination rate than black seeds at all temperature and salinity treatments. The highest rate of germination of both seeds occurred at the temperature regime of 5°-15°C. Recovery of germination for black seeds when transferred to distilled water after being in various salinity treatments for 20 days was quite variable. Recovery decreased with increase in salinity at lower temperature regimes, increased with salinity at optimal thermoperiod, and had no effect at 20°-30°C. Brown seeds recovered poorly from salinity at all thermoperiods except 5°-15°C, where recovery decreased with an increase in salinity. Brown seeds are adapted to germination in the early part of the growing season, whereas black seeds are capable of surviving harsher conditions and can germinate in later time periods. Characteristics of the dimorphic seeds increase chances for survival in the harsh saline desert environment.     

Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor.

The T-cell receptor is necessary and sufficient for recognition of peptides presented by major histocompatibility complex molecules. Other adhesion molecules, like CD4 or CD8, play an auxiliary role in antigen recognition by T cells. Here we analyse T-cell receptor (TCR) binding using a soluble rather than a cell-bound receptor molecule. A TCR-immunoglobulin chimaera is constructed with the variable and the first constant regions of both the TCR alpha- and beta-chains linked to the immunoglobulin light-chain constant regions. This soluble TCR is expressed, assembled and secreted as an alpha beta heterodimer by a myeloma cell line transfected with the recombinant genes. Furthermore, the soluble TCR is biologically active: it specifically inhibits antigen-dependent activation of the relevant T-cell clones and thus discriminates between proper and irrelevant peptides presented by major histocompatibility complex molecules.     

A new method of scenario analysis for strategic planning

We first present scenario analysis as a qualitative forecasting technique useful for strategic planning. Then we develop an overview of the two classes of methods for scenario analysis described in the literature. Based on both classes, a new method is developed which especially fits the needs of strategic planning. The method can be divided into three stages: 1. Determination of compatible scenarios, 2. Determination of scenario probabilities, and 3. Determination of main scenarios. An example is given to illustrate the method.     

Einfluss von angiotensin auf das argentaffine system des meerschweinchens

Summary The duodenum of guinea-pigs were dissected immediately after death and incubated for 10 min at 37 °C in a solution of Krebs-Ringer-Bicarbonate buffer, in which Hypertensin (CIBA) was added. The result was that the argentaffine cells were more numerous after the incubation period, as compared with the controls. The 2 controls were pieces placed into the solutions of Formalin and blank Krebs-Ringer-Bicarbonate buffer.