The current through the L‐type calcium channel is inhibited and stimulated by distinct dihydropyridines at very low concentrations. The molecular determinants for the high affinity block and stimulation were investigated using chimeras between the class C and E calcium channels. Mutation of three amino acids in the last putative transmembrane segment (IVS6) of the alpha1C subunit decreased the affinity for (+)isradipine 100‐fold without significantly affecting the basic properties of the expressed channel. Mutation of two of these three amino acids completely abolished the stimulatory effect of the calcium channel agonist Bay K 8644. These mutations only slightly affected the blocking efficacy of mibefradil and the phenylalkylamine devapamil. Three distinct but adjacently located amino acids mediated the high affinity block by devapamil. These results suggest that the IVS6 segment of the alpha1C subunit is critical for the high affinity interaction between the L‐type calcium channel and the calcium channel agonist Bay K 8644 and the two antagonists isradipine and devapamil.

The Ca2+ channel subunits alpha 1C-a and alpha 1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba2+ current (IBa) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 microM cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 microM PKA and 1 microM okadaic acid. The activity of the alpha 1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 microM H 89 and was not increased by superfusion with 5 microM forskolin plus 20 microM isobutyl-methylxanthine (IBMX). The alpha 1C-a.beta 2.alpha 2/delta complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 microM H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the alpha 1C-a.beta 2.alpha 2/delta channel with 10 microM PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca2+ current (ICa) of cardiac myocytes increased threefold during internal dialysis with 5 microM PKA or 25 microM microcystin and during external superfusion with 0.1 microM isoproterenol or 5 microM forskolin plus 50 microM IBMX. These results indicate that the L-type Ca2+ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.

No abstract

Integrins of the ␤7 subfamily, ␣4␤7 and ␣E␤7, contribute to lymphocyte homing and to the development of protective or autoreactive immune responses at mucosal sites. The ␤ subunits of integrins are considered important for regulation of stimulated cell adhesion and adhesion-dependent signal transduction. Using a yeast interaction trap screen, a human WD repeat protein, termed WAIT-1, was isolated that interacts with the integrin ␤7 cytoplasmic tail and is homologous to mouse EED and Drosophila ESC proteins. WAIT-1 also binds to the cytoplasmic domains of ␣4 and ␣E but not to those of integrin ␤1, ␤2, and ␣L subunits. Association of WAIT-1 and ␤7-integrin was confirmed by coprecipitation from transiently transfected 293 cells. The binding site for WAIT-1 was mapped to a short membrane-proximal region of the ␤7 cytoplasmic tail with Tyr-735 being of critical importance. Northern blot analysis revealed multiple WAIT-1-related transcripts with differential expression in circulating leukocytes, tissue-resident cells of diverse origin, and lymphoid malignancies. These results suggest that WAIT-1, together with the recently identified RACK1, may define a novel subfamily of WD repeat proteins that interact with distinct subsets of integrin cytoplasmic tails and may act as specific regulators of integrin function.Expression of ␤7-integrins, ␣4␤7 and ␣E␤7, is largely confined to leukocytes. Although ␣4␤7-integrin is homogeneously expressed on natural killer cells, eosinophils, and naive T and B cells, its distribution on effector/memory T and B cells is restricted to a subset of gut-homing lymphocytes (1-6). Peripheral blood monocytes do not express ␤7-integrins, but ␣4␤7 and ␣E␤7 are up-regulated upon stimulation of macrophage differentiation with phorbol ester or interferon-␥ (7). Recent studies have indicated that, in addition, ␣4␤7-integrin is induced on endothelial cells after treatment with proinflammatory mediators such as tumor necrosis factor (8). In contrast to ␣4␤7, expression of the ␣E␤7-integrin is confined to a subset of gutassociated T lymphocytes and dendritic cells (4,6,9).␤7-Integrins are considered important for the development and function of gut-associated lymphoid tissues. Interaction of ␣4␤7 with MAdCAM-1 allows for tissue-specific migration of circulating lymphocytes into the lamina propria and Peyer's patches of the gut (10, 11), whereas ␣E␤7 may retain intraepithelial lymphocytes within the gut epithelium through binding of E-cadherin on epithelial cells (12,13). Lack of ␤7-integrins severely impairs the development of the gut immune system, as Peyer's patches are absent or hypoplastic, and fewer intraepithelial lymphocytes are detected in ␤7-integrin-deficient mice (14). Moreover, gut-homing ␣4␤7 ϩ CD4 T cells specifically harbor cellular memory for intestinal antigens, suggesting that ␣4␤7 helps to target and segregate intestinal versus systemic immune responses (15).Integrins of the ␤7 family are involved in the development and/or progression of diseases such as colitis (16,17), diabetic insul...

Interactions of the integrins alpha(4)beta(7) with its cognate ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) play a crucial role in the development of mucosa-associated lymphoid organs, in the generation of mucosal immune responses, and in diverse pathological processes such as chronic inflammatory bowel disease and type I diabetes. Using a previously developed spatial screening technique we describe the development of potent and selective alpha(4)beta(7) integrin antagonists based on the domain 1 Leu-Asp-Thr (LDT) sequence of MAdCAM-1 that is essential for alpha(4)beta(7) integrin binding. A library of homodetic cyclic penta- and hexapeptides was synthesized presenting the pharmacophoric LDT-sequence in different conformations. The cyclic hexapeptide P10 cyclo(Leu-Asp-Thr-Ala-D-Pro-Ala) inhibits alpha(4)beta(7) integrin mediated cell adhesion to MAdCAM-1 effectively. Further optimization of the lead structure P10 resulted in cyclic hexapeptides with enhanced activity. The compounds P25 cyclo(Leu-Asp-Thr-Ala-D-Pro-Phe), P28 cyclo(Leu-Asp-Thr-Asp-D-Pro-Phe), P29 cyclo(Leu-Asp-Thr-Asp-D-Pro-His), and P30 cyclo(Leu-Asp-Thr-Asp-D-Pro-Tyr) strongly inhibited alpha(4)beta(7) integrin mediated cell adhesion to MAdCAM-1, but they did not affect binding of the closely related alpha(4)beta(1) integrin to VCAM-1.

As part of our ongoing research in the development of alpha4beta7 integrin antagonists, we are interested in peptidomimetics based on a rigid scaffold to allow the display of essential side chains in a suitable binding conformation while eliminating backbone amide bonds and therefore improving pharmacokinetic parameters of the drug. Except for a few examples, peptidomimetics scaffolds have only been moderately successful and often yield molecules that lack the potency of their peptide counterparts. However, we present herein a successful application of using a rigid scaffold. Starting from a mannopyranoside analogue previously discovered in our laboratory as an inhibitor of the alpha4beta1/vascular cell adhesion molecule interaction, a biased library of functionalized carbohydrates was developed. One compound emerged from this library as an active and selective antagonist toward the alpha4beta7/mucosal addressin cell adhesion molecule interaction. Conformational implications and the relevance of different pharmacophoric patterns will be discussed in order to explain the reverse selectivity and enhanced affinity.

The diheme cytochromes c of the widespread TsdA family are bifunctional thiosulfate dehydrogenase/tetrathionate reductases. Here, biochemical information was collected about TsdA from the Epsilonproteobacterium Wolinella succinogenes (WsTsdA). The situation in W. succinogenes is unique since TsdA is closely associated with the unprecedented lipoprotein TsdC encoded immediately downstream of tsdA in the same direction of transcription. WsTsdA purified from Escherichia coli catalyzed both thiosulfate oxidation and tetrathionate reduction. After co-production of TsdC and WsTsdA in E. coli, TsdC was found to mediate membrane attachment of TsdA and to ensure its full catalytic activity. This effect was much stronger in the tetrathionate-reducing than in the thiosulfate-oxidizing direction. It is concluded that the TsdAC complex predominantly acts as a tetrathionate reductase in vivo.

Ein cyclisches Peptid diente als Vorbild für das Design und die Synthese einer neuen Klasse von biologisch aktiven und α4‐selektiven Integrinantagonisten (z.B. 1) auf der Grundlage von β‐D‐Mannose. Diese auf Kohlenhydraten basierenden Peptidmimetika enthalten die funktionellen Gruppen ihrer cyclischen Peptidvorstufen, aber nicht das in diesem Fall überflüssige cyclische Rückgrat an Amidbindungen.