Subthreshold-activating somatodendritic A-type potassium channels have fundamental roles in neuronal signaling and plasticity which depend on their unique cellular localization, voltage dependence, and kinetic properties. Some of the components of A-type K(+) channels have been identified; however, these do not reproduce the properties of the native channels, indicating that key molecular factors have yet to be unveiled. We purified A-type K(+) channel complexes from rat brain membranes and found that DPPX, a protein of unknown function that is structurally related to the dipeptidyl aminopeptidase and cell adhesion protein CD26, is a novel component of A-type K(+) channels. DPPX associates with the channels' pore-forming subunits, facilitates their trafficking and membrane targeting, reconstitutes the properties of the native channels in heterologous expression systems, and is coexpressed with the pore-forming subunits in the somatodendritic compartment of CNS neurons.

We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-α-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.

The underivatized saponins from Tribulus terrestris and Panax ginseng have been investigated by electrospray ionization multi-stage tandem mass spectrometry (ESI-MS n ). In ESI-MS spectra, a predominant [M ϩ Na] ϩ ion in positive mode and [M Ϫ H] Ϫ ion in negative mode were observed for molecular mass information. Multi-stage tandem mass spectrometry of the molecular ions was used for detailed structural analysis. Fragment ions from glycoside cleavage can provide information on the mass of aglycone and the primary sequence and branching of oligosaccharide chains in terms of classes of monosaccharides. Fragment ions from cross-ring cleavages of sugar residues can give some information about the linkages between sugar residues. It was found that different alkali metal-cationized adducts with saponins have different degrees of fragmentation, which may originate from the different affinity of a saponin with each alkali metal in the gas phase. ESI-MS n has been proven to be an effective tool for rapid determination of native saponins in extract mixtures, thus avoiding tedious derivatization and separation steps. (J Am Soc Mass Spectrom 2004, 15, 133Ϫ141)

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membranebound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPIanchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.The Epidermal Growth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) 2 family of genes, including mammalian Cripto-1 (CR-1/Tdgf1), has been shown to play an important role in vertebrate development and in tumor progression (1). EGF-CFC proteins are indispensable for early embryonic development, being involved in the formation of the primitive streak, patterning of the anterior/posterior axis, mesoendoderm formation, and establishment of left-right asymmetry (2). CR-1 is an oncofetal gene, which is expressed during early developmental stages and is re-expressed in several types of human carcinomas (3). In fact, CR-1 protein is highly expressed in a number of human carcinomas, including breast (75-82%), colon (67-84%), and gastric cancer (33-47%), where it is being assessed as a tumor-specific target for immunotherapy (3, 4). Mice that overexpress a human CR-1 transgene selectively in mammary epithelium develop mammary hyperplasia and adenocarcinomas (5-7). Conversely, blockade of CR-1-mediated signaling suppresses tumor growth (4, 8, 9).CR-1 functions through at least three different signaling pathways: 1) as a co-receptor for the transforming growth factor ␤-related proteins Nodal and growth and differentiation factors 1 and 3 (10 -12), 2) as a ligand for glypican-1/c-Src/ MAPK/PI3K-Akt signaling (13), and 3) as an inhibitor for activin/transforming growth factor-␤ signaling (8,14). Nodal requires EGF-CFC proteins as co-receptors to bind the activin type I receptors (activin-like kinases 4 and 7) and activin type II receptor (ActRII). Embryological defects in CR-1-null mice are lethal mainly due to a disruption of Nodal-dependent signaling (15). CR-1 can also function independently of Nodal as a ligand for glypican-1, which can act...

The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (Kd ∼100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment.