The vacuolar-ATPase B1 subunit in distal tubular acidosis: novel mutations and mechanisms for dysfunction

Mutations in the B1 subunit of the multisubunit vacuolar ATPase cause autosomal-recessive distal renal tubular acidosis and sensorineural deafness. Here, we report a novel frameshift mutation that truncates the C-terminus of the human B1 subunit. This mutant protein failed to assemble with other subunits in the cytosol to form the complex that can be targeted to vesicular structures in mammalian cells. Loss of proton pump activity was demonstrated in a functional complementation assay in B-subunit null yeast. The mutation caused loss of a discreet C-terminal region critical for subunit interaction not related to the C-terminal PDZ motif. Co-expression studies failed to demonstrate dominant negative effects of this truncated mutant over wild-type B1. Analysis of 12 reported B1 subunit missense mutations showed one polymorphic allele had intact pump function, two point mutants had intact assembly but defective proton pumping, and the remaining nine had disrupted assembly with no pump function. One presumed polymorphic allele was actually an inactivating mutation. Our study shows that multiple mechanisms of pump dysfunction result from B1 subunit mutations with a common outcome being defective assembly. Polymorphisms of the B1 subunit in the general population may affect renal acidification and urinary chemistry.

Bone mass gain after parathyroidectomy

Yajima and co-workers investigated iliac bone biopsies taken before and after parathyroidectomy. They found enhanced de novo osteoid formation and mineral apposition at trabecular sites without signs of previous bone resorption. From this finding they conclude that 'minimodeling' contributes to the increase of bone volume following parathyroidectomy. This report refines our understanding of the compensatory mechanisms by which bone mass and possibly increased mechanical stability of the skeletal apparatus are regained after parathyroidectomy.

Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.

Renal safety of annual zoledronic acid infusions in osteoporotic postmenopausal women

Intravenous bisphosphonates reduce fracture risk but have been associated in rare cases with deteriorating renal-function in cancer patients. The renal effects of zoledronic acid were assessed in osteoporotic postmenopausal women from 27 countries who received three annual infusions of zoledronic acid or a placebo in a randomized, double-blind trial. Serum creatinine, estimated creatinine clearance and urinary protein were measured before and after at least one infusion in a predefined renal safety cohort of 5035 equally divided patients. This group was compared to 7714 patients whose parameters were measured annually. Significantly more transient pre- to post-infusion increases in serum creatinine occurred in zoledronic acid than placebo-treated patients with significant elevations, relative to pre-infusion, only in the second year. All 31 zoledronic acid and 8 of 10 patients on placebo recovered their pre-infusion serum creatinine value within 12 months. No differences in mean changes in serum creatinine, estimated creatinine clearance or adverse renal events were found. We found that transient changes in renal function can occur following an annual zoledronic acid infusion but, in the long term, renal function was not different from control patients.

Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

In silico target fishing for rationalized ligand discovery exemplified on constituents of Ruta graveolens

The identification of targets whose interaction is likely to result in the successful treatment of a disease is of growing interest for natural product scientists. In the current study we performed an exemplary application of a virtual parallel screening approach to identify potential targets for 16 secondary metabolites isolated and identified from the aerial parts of the medicinal plant RUTA GRAVEOLENS L. Low energy conformers of the isolated constituents were simultaneously screened against a set of 2208 pharmacophore models generated in-house for the IN SILICO prediction of putative biological targets, i. e., target fishing. Based on the predicted ligand-target interactions, we focused on three biological targets, namely acetylcholinesterase (AChE), the human rhinovirus (HRV) coat protein and the cannabinoid receptor type-2 (CB (2)). For a critical evaluation of the applied parallel screening approach, virtual hits and non-hits were assayed on the respective targets. For AChE the highest scoring virtual hit, arborinine, showed the best inhibitory IN VITRO activity on AChE (IC (50) 34.7 muM). Determination of the anti-HRV-2 effect revealed 6,7,8-trimethoxycoumarin and arborinine to be the most active antiviral constituents with IC (50) values of 11.98 muM and 3.19 muM, respectively. Of these, arborinine was predicted virtually. Of all the molecules subjected to parallel screening, one virtual CB (2) ligand was obtained, i. e., rutamarin. Interestingly, in experimental studies only this compound showed a selective activity to the CB (2) receptor ( Ki of 7.4 muM) by using a radioligand displacement assay. The applied parallel screening paradigm with constituents of R. GRAVEOLENS on three different proteins has shown promise as an IN SILICO tool for rational target fishing and pharmacological profiling of extracts and single chemical entities in natural product research.

Glycyrrhetinic acid food supplementation lowers serum potassium concentration in chronic hemodialysis patients

Hyperkalemia is a common life-threatening problem in hemodialysis patients. Because glycyrrhetinic acid (GA) inhibits the enzyme 11beta-hydroxy-steroid dehydrogenase II and thereby increases cortisol availability to the colonic mineralocorticoid receptor, it has the potential to lower serum potassium concentrations. To test this, 10 patients in a 6 month prospective, double-blind, placebo-controlled crossover study were given cookies or bread rolls supplemented with glycyrrhetinic acid or placebo. Twenty-four-hour blood pressure measurements were performed at baseline and week 6 and 12 of each treatment period. The ratio of plasma cortisol/cortisone was significantly increased in all patients on GA as compared to baseline or placebo, indicating appropriate enzyme inhibition. Nine of the 10 patients had a persistent decrease in predialysis serum potassium concentration. On GA, mean predialysis serum potassium was significantly lower than at baseline or on placebo. On placebo, serum potassium was significantly elevated above the upper limit of normal in 76% compared to 30% of measurements during GA treatment. Furthermore, on this treatment the frequency of severe hyperkalemia significantly decreased from 9% to 0.6%. No differences were found in parameters reflecting sodium retention. Although these studies show that prolonged GA supplementation persistently lowers serum potassium in dialysis patients, a long-term toxicity study will be mandatory before we recommend the routine use of this treatment.

LogoTakt - hochfrequente, getaktete Belieferung in kleinen Sendungseinheiten

Innerhalb des im Rahmen des Projektes LogoTakt (BMWi, Förderkennzeichen 19 G 7036 E) entwickelten Transportkonzeptes für Großladungsträger wird zwischen Versendern und Empfängern eine hochfrequente Taktung eingeführt, die zu einer Reduktion der Bestände und höherer Zuverlässigkeit führt. Um zu vermeiden, dass durch die höhere Frequenz die Transportkosten steigen, müssen die Transporte in einem offenen Netzwerk mehrerer Teilnehmer stattfinden und die so entstehenden Konsolidierungseffekte genutzt werden. Im Projekt LogoTakt wurde für ein solches Netzwerk ein Anreiz- und Vergütungsmodell entwickelt, das die Zusammenarbeit von Versendern, Verladern, Spediteuren und Empfängern regelt. Durch die erhöhte Zuverlässigkeit entsteht trotz höherer Frequenz weniger Dispositionsaufwand, da die Transporte auf strategischer Ebene geplant werden können. Weiterhin wird die Disposition durch ein Abweichungsmanagement unterstützt, das alle laufenden Transporte überwacht und bei Bedarf aus der Historie mögliche Lösungen für die Abweichung vorschlägt. Diese KI-Komponente sorgt gleichzeitig durch eine ständige Analyse der historischen Daten für einen strukturierten kontinuierlichen Verbesserungsprozess, der die operative mit der strategischen Ebene verbindet. LogoTakt ermöglicht durch die Integration strategischer und operativer Komponenten zu einem Softwaretool eine bessere Planbarkeit eines komplexen Transportsystems unter Einbeziehung von Lean-Erkenntnissen. Letztendlich können durch dieses Konzept gleichzeitig Ziele, die häufig im Bezug auf Green Logitics diskutiert werden, wie beispielsweise CO2-Einsparungen oder die Reduktion von Straßenkilometern, erreicht werden.

Palaeohydrology, fires and vegetation succession in the southern Baltic during the last 7500 years reconstructed from a raised bog based on multi-proxy data

We present the first 7500 yr long multi-proxy record from a raised bog located at the southern Baltic coast, Poland. Testate amoebae, plant macrofossils, pollen and microscopic charcoal were used to reconstruct environmental changes in Pomerania (northern Poland, Kaszuby Lakeland) from a 7-m thick peat archive of Stążki bog dated 5500 BC–AD 1250. We obtained a record of proxies representing different spatial scales: regional vegetation changed simultaneously with local vegetation, and testate amoebae showed a pattern of change similar to that of pollen and plant macrofossils. On the basis of the combined proxies, we distinguished three hydroclimatic stages: moist conditions 5500–3450 BC, drier conditions with regionally increased fires up to 600 BC, and again moist conditions from 600 BC onward. During the drier interval, a first climatic shift to wetter conditions at 1700 BC is indicated by regional pollen as the replacement of Corylus by Carpinus, and locally by, e.g., the increase of Hyalosphenia elegans and mire plants such as Sphagnum sec. Cuspidata. Furthermore, we observed a correlation since 600 BC among the re-expansion of Carpinus (after a sudden decline ca. 950 BC), increased peat accumulation, increase of Hyalosphenia species, and fewer fires, suggesting lower evapotranspiration and a stable high water table in the bog. Fagus started to expand after AD 810 gradually replacing Carpinus, which was possibly due to a gradually more oceanic climate, though we cannot exclude human impact on the forests. Peat accumulation, determined by radiocarbon dating, varied with bog surface wetness. The hydroclimatic phases found in Stążki peatland are similar to moisture changes recorded in other sites from Poland and Europe. This is the first detailed record of hydroclimatic change during the Holocene in the southern Baltic region, so it forms a reference site for further studies on other southern Baltic bogs that are in progress.

Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

EGFR exon-level biomarkers of the response to bevacizumab/erlotinib in non-small cell lung cancer

Activating epidermal growth factor receptor (EGFR) mutations are recognized biomarkers for patients with metastatic non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs can also have activity against NSCLC without EGFR mutations, requiring the identification of additional relevant biomarkers. Previous studies on tumor EGFR protein levels and EGFR gene copy number revealed inconsistent results. The aim of the study was to identify novel biomarkers of the response to TKIs in NSCLC by investigating whole genome expression at the exon-level. We used exon arrays and clinical samples from a previous trial (SAKK19/05) to investigate the expression variations at the exon-level of 3 genes potentially playing a key role in modulating treatment response: EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and vascular endothelial growth factor (VEGFA). We identified the expression of EGFR exon 18 as a new predictive marker for patients with untreated metastatic NSCLC treated with bevacizumab and erlotinib in the first line setting. The overexpression of EGFR exon 18 in tumor was significantly associated with tumor shrinkage, independently of EGFR mutation status. A similar significant association could be found in blood samples. In conclusion, exonic EGFR expression particularly in exon 18 was found to be a relevant predictive biomarker for response to bevacizumab and erlotinib. Based on these results, we propose a new model of EGFR testing in tumor and blood.

Politique étrangère suisse

Le conseiller fédéral Didier Burkhalter a pris la direction du Département fédéral des affaires étrangères (DFAE). – Le parlement a octroyé un crédit de 11.35 milliards de francs pour la coopération internationale 2013-2016. – Le Conseil fédéral a activé la clause de sauvegarde envers les Etats de l’UE-8. – Les questions institutionnelles ont continué à bloquer les relations bilatérales avec l’UE. – L’Allemagne et les Etats-Unis ont maintenu la pression sur la place financière suisse lors des négociations d’accords de double-imposition. – Le peuple a refusé l’initiative de l’ASIN « La parole au peuple ! ». – La Suisse a pris position sur le conflit syrien en instaurant des sanctions contre le régime. – La Suisse a fêté ses 10 ans d’adhésion à l’ONU et a reçu son secrétaire général Ban Ki-Moon. – La Suisse a accueilli à Berne le Prix Nobel de la Paix Aung San Suu Kyi et a ouvert une ambassade au Myanmar.