TWIK1 belongs to a family of K þ channels involved in neuronal excitability and cell volume regulation. Its tissue distribution suggests a role in epithelial potassium transport. Here we show that TWIK1 is expressed in a subapical compartment in renal proximal tubules and in polarized MDCK cells. In nonpolarized cells, this compartment corresponds to pericentriolar recycling endosomes. We identified EFA6, an exchange factor for the small G protein ADP-ribosylation factor 6 (ARF6), as a protein binding to TWIK1. EFA6 interacts with TWIK1 only when it is bound to ARF6. Because ARF6 modulates endocytosis at the apical surface of epithelial cells, the ARF6/EFA6/TWIK1 association is probably important for channel internalization and recycling.

The mechanisms governing vascular smooth muscle tone are incompletely understood. In particular, the role of the sarcolemmal calcium pump PMCA (plasma membrane calmodulin-dependent calcium ATPase), which extrudes Ca 2؉ from the cytosol, and its importance compared with the sodium/calcium exchanger remain speculative. To test whether the PMCA is a regulator of vascular tone, we generated transgenic mice overexpressing the human PMCA4b under control of the arterial smooth muscle-specific SM22␣ promoter. This resulted in an elevated systolic blood pressure compared with littermate controls. In PMCA-overexpressing mice, endothelium-dependent relaxation of norepinephrine-preconstricted aortic rings to acetylcholine did not differ from wild type controls (76 ؎ 8% versus 79 ؎ 8% of maximum relaxation; n ‫؍‬ 12, n.s.). De-endothelialized aortas of transgenic mice exhibited stronger maximum contraction to KCl (100 mmol/liter) compared with controls (86 ؎ 6% versus 68 ؎ 7% of reference KCl contraction at the beginning of the experiment; p <0.05). Preincubation of de-endothelialized vessels with the nitric oxide synthase (NOS) inhibitor L-NAME (L-N(G)-nitroarginine methyl ester) (10؊5 mol/liter) resulted in a stronger contraction to KCl (p <0.05 versus without L-NAME), thus unmasking vasodilatory effects of inherent NO production. Maximum contraction to KCl after preincubation with L-NAME did not differ between PMCA mice and controls. In analogy to the results in PMCAoverexpressing mice, contractions of de-endothelialized aortas of neuronal NOS-deficient mice to KCl were significantly increased compared with controls (151 ؎ 5% versus 131 ؎ 6% of reference KCl contraction; p <0.05). In conclusion, our data suggest a model in which the sarcolemmal Ca 2؉ pump down-regulates activity of the vascular smooth muscle Ca 2؉ /calmodulin-dependent neuronal NOS by a functionally relevant interaction. Therefore, the PMCA represents a novel regulator of vascular tone.

An overview of anticancer active spirocyclopropanes of the illudin class is provided. After a short introduction on the history and general chemistry of illudins M and S, new discoveries concerning their mode of action and metabolism are reported as well as new synthetic endeavors towards derivatives with improved selectivity for and efficacy against cancer cells. In addition, common and recently tapped biological sources and isolation procedures for known and new illudins are discussed. Pertinent literature is covered up to 2010.

New combretastatin A analogues featuring oxazole or N-methylimidazole bridged Z-alkenes and halo- or amino-substituted A-rings were tested against various cancer cell lines and in testicular germ cell tumor xenografts in mice. Imidazoles with 3-halo-4,5-dimethoxy substituted A-rings and 3-amino-4-methoxy substituted B-rings (7b and 8b) were efficacious at nanomolar concentrations against cells of combretastatin A refractory HT-29 colon carcinoma, multidrug-resistant MCF-7/Topo breast carcinoma, and cisplatin-resistant 1411HP testicular germ cell tumor. They induced apoptosis and inhibited tubulin polymerization. While well tolerated by mice at high doses, these imidazoles initiated extensive intratumoral hemorrhage and regressions of highly vascularized 1411HP xenografts.

The unfavorable therapeutic index of the fungal cytotoxin illudin M was to be improved by covalent attachment of the redox modulator and phenyl isobiostere ferrocene. Esters of illudin M with ferrocenoic and 1,1'-ferrocenedioic acid were prepared, structurally characterised (X-ray), and tested for cytotoxicity [MTT assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], induction of apoptosis (TUNEL assay; western blotting for caspase-9), and tumor specificity in cells of human HL-60 leukemia, human 518A2 melanoma, and in nonmalignant human foreskin fibroblasts. The diester of illudin M with 1,1'-ferrocenedioic acid was distinctly more antiproliferative and apoptosis inducing in the melanoma cells [half maximal inhibitory concentration, IC50(48 h) = 0.4+/-0.1 micromol/l] than in the HL-60 cells [IC50(48 h) = 3.0+/-1.6 micromol/l] and in the nonmalignant fibroblasts [IC50(48 h) = 3.7+/-1.9 micromol/l]. This corresponds to a doubling of the therapeutic index with respect to illudin M. The monoester of illudin M with ferrocenoic acid was nine times less efficacious in the cancer cells, when compared with the diester. In conclusion, the ferrocene diminishes the general toxicity of the illudin M moiety and increases its cell line specificity. The bis(illudinyl M) 1,1'-ferrocenedioate presumably operates by a synergistic, two-pronged attack on its molecular targets.