Inhibition of palmitate‐induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6)

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta‐cells, an event linked to apoptotic loss of β‐cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage‐inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin‐producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA‐activated protein kinase‐like ER‐localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate‐induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin‐producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Endothelial-Mesenchymal Transition of Brain Endothelial Cells: Possible Role during Metastatic Extravasation

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.     

Determination of high-affinity oestrogen-receptor sites in uterine supernatant preparations

An assay method was developed for the determination of high-affinity oestradiol receptors in uterine supernatant preparations. When only high-affinity sites are present in such preparations, or when they predominate, the analysis of the equilibrium between oestradiol and receptor sites based on the Scatchard (1949) plot may be used to determine the dissociation constant of the equilibrium and the molar concentration of the high-affinity sites. When both high-affinity and low-affinity sites are present the Scatchard plot is no longer linear and cannot be used directly to determine high-affinity sites. Determination of the reverse velocity constants of the reaction between high-affinity (k(-1)) and low-affinity (k(-2)) receptor sites and [(3)H]oestradiol has shown that these constants differ by at least one order of magnitude. Advantage has been taken of this difference to introduce an additional step into the assay procedure that eliminates oestradiol bound to low-affinity sites and permits the determination of high-affinity sites in different species and under a variety of physiological conditions.     

Versuch einer Kausalanalyse der geotropischen Reaktionskette im Chara-Rhizoid

Summary In the initial phase of the geotropical reaction of the Chara rhizoid the growth difference postulated by Sievers (1967c) between the physically upper, slightly subapical flank and the lower one is demonstrated. In horizontal exposure the growth of the extreme cell apex is continued, while the growth of the lower flank is inhibited and that of the upper one is promoted. In the end phase the cell apex shows a damped oscillation until it finally reaches the vertical growth direction. The statoliths follow the oscillating growth of the cell tip from one flank to the opposite one until they are statistically equally redistributed in their normal position.—In vertical exposure under reduced turgor pressure the statoliths fall down into the extreme cell apex, where they inhibit the growth of this part of the cell wall, while the subapical wall grows transversally.—It is concluded that the statoliths inhibit the growth of the cell wall area which they cover.—The physical phase of the reaction chain, the susception, is the gravity-induced downward displacement of the statoliths. The physiological phase starts with the diversion of the acropetal transport of the Golgi vesicles to the upper part of the cell, which is caused by the block of statoliths (perception). The greater rate of vesicle incorporation into the upper flank in comparison to the lower one causes the subapical growth difference which results in the curvature (reaction).—In the case of the Chara rhizoid Golgi- and statolith-apparatus function as a self-regulating cellular system.

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Herrn Prof. Dr. Dr. h. c. Kurt Mothes zum 70. Geburtstag.     

Effects of ditch management on caddisfly, dragonfly and amphibian larvae in intensively farmed peat areas

Conservation of natural values within farming practice is growing rapidly within the Netherlands. The focus is primarily on terrestrial flora and fauna such as the vegetation in ditch banks and meadow birds. Knowledge needed to enhance biodiversity in ditches is limited. Therefore, a field study was set up to determine the effects of dredging, ditch cleaning and nutrient supply in the adjacent fields on caddisfly, dragonfly and amphibian larvae in the ditches in a peat area.Two-hundred forty ditches spread over 84 dairy farms were selected to determine the individual effect of several management aspects. Generalised linear modelling was used as a tool to detect the most relevant aspects and to obtain quantitative relations with the chance of the larvae being present.Dredging had an impact on the presence of all larvae types. The type of dredging machine, the dredging period, the water depth and the frequency of dredging can influence the presence of the larvae. The presence of caddisfly larvae was also affected by the cleaning machine and period and by the P supply in the adjacent field. The presence of amphibian larvae was also affected by the cleaning period.Measures that will enhance the presence of the larvae are formulated. Options for water boards and other government authorities to stimulate farmers to take these measures are given.     

End Stage Renal Disease Predicts Increased Risk of Death in First Degree Relatives in the Norwegian Population

BackgroundIncreased risk of end stage renal disease (ESRD) and death in Norwegian living kidney donors has been reported, most of the donors were related to the recipient. The present study investigates risk of death in first degree relatives of ESRD patients.MethodsThe Norwegian Population Registry, The Norwegian Cause of Death Registry and the Norwegian Renal Registry were linked. All citizens born in Norway, alive in 1960 and with at least one registered first degree relative were included; individuals who died during the first year of life were excluded. A cohort-design was used, ESRD in a first degree relative was the main exposure variable and death and causes of death were the main outcome variables. Cox regression statistics were used to investigate mortality risks.Results5 130 600 individuals were included, 27 508 had at least one first degree relative with ESRD. 828 022 died during follow-up, of whom 4105 had a first degree relative with ESRD. Adjusted hazard ratio (aHR) for death was 1.13 (1.09–1.16) in individuals with a relative with ESRD compared to those without a relative with ESRD. Excluding known hereditary renal disease, aHR decreased to 1.12 (1.09–1.15). Cardiovascular death aHR was 1.15 (1.10–1.21), of which cerebrovascular death 1.34 (1.22–1.50). aHR for death due to non-hereditary renal/ureteric disease was 2.29 (1.81–2.91) with renal failure 1.80 (1.26–2.56) and glomerular disease 5.69 (3.88–8.34) as main contributors. Diabetes mellitus death aHR was 1.68 (1.35–2.10). Absolute mortality risks increased most for the oldest cohorts with excess mortality of 148 per 100.000 person years for the cohort born 1920–39 and 218 for the cohort born 1900–1919.ConclusionsESRD in first degree relatives was associated with increased hazard ratio for death. Death due to cardiovascular disease, renal disease and diabetes mellitus increased the most.