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Helicobacter pylori persistently colonize the human stomach and have been linked to atrophic gastritis and gastric carcinoma. Although it is well known that H. pylori infection can result in hypochlorhydria, the molecular mechanisms underlying this phenomenon remain poorly understood. Here we show that VacA permeabilizes the apical membrane of gastric parietal cells and induces hypochlorhydria. The functional consequences of VacA infection on parietal cell physiology were studied using freshly isolated rabbit gastric glands and cultured parietal cells. Secretory activity of parietal cells was judged by an aminopyrine uptake assay and confocal microscopic examination. VacA permeabilization induces an influx of extracellular calcium, followed by activation of calpain and subsequent proteolysis of ezrin at Met(469)-Thr(470), which results in the liberation of ezrin from the apical membrane of the parietal cells. VacA treatment inhibits acid secretion by preventing the recruitment of H,K-ATPase-containing tubulovesicles to the apical membrane of gastric parietal cells. Electron microscopic examination revealed that VacA treatment disrupts the radial arrangement of actin filaments in apical microvilli due to the loss of ezrin integrity in parietal cells. Significantly, expression of calpain-resistant ezrin restored the functional activity of parietal cells in the presence of VacA. Proteolysis of ezrin in VacA-infected parietal cells is a novel mechanism underlying H. pylori-induced inhibition of acid secretion. Our results indicate that VacA disrupts the apical membrane-cytoskeletal interactions in gastric parietal cells and thereby causes hypochlorhydria.
Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK(rat5aa) cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK(human5aa) cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK(rat5aa) cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK(rat5aa) cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na(+) in MDCK(rat5aa) cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca(2+)-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK(rat5aa) cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK(rat5aa) cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.
Polydisperse Ficoll mixtures have been used to explore glomerular sieving. Ficoll appears to be neither absorbed nor secreted by the renal tubule, and so urinary Ficoll concentrations reflect only the glomerular filtration barrier. The literature is contradictory regarding Ficoll's behavior as an idealized spherical solute. Further definition of Ficoll transport will inform interpretation of in vivo results. Flat-sheet membranes comprising a uniform array of slit pores measuring 8 nm by 45 mum were perfused with FITC-labeled Ficoll 70 and BSA. Ficoll and BSA concentrations were quantified by gel-permeation chromatography and Bradford assay, respectively. BSA and Ficoll molecules with diameters equal to approximately half of the slit pore width displayed hindered transport in agreement with modeled rigid sphere transport through slit-shaped pores. Ficoll molecules larger than approximately 0.65 slit width displayed transport rates in excess of predictions. Ficoll molecules with Stokes-Einstein diameters greater than the pore dimension were observed in permeate samples. We present data for Ficoll filtration through a novel array of well-defined pores, which illustrate that Ficoll is well modeled as an ideal sphere in one size domain, but the model breaks down as molecular diameter approaches pore size. These data inform the present debate regarding glomerular filtration and affect conclusions drawn from the use of Ficoll as a tracer molecule. The apparent hyperpermeability of Ficoll through slit-shaped pores suggests that further modeling incorporating deformation of the molecule is necessary when using Ficoll solutions to characterize membranes.
BACKGROUND - An interlocked transcriptional-translational feedback loop (TTFL) is thought to generate the mammalian circadian clockwork in both the central pacemaker residing in the hypothalamic suprachiasmatic nuclei and in peripheral tissues. The core circadian genes, including Period1 and Period2 (Per1 and Per2), Cryptochrome1 and Cryptochrome2 (Cry1 and Cry2), Bmal1, and Clock are indispensable components of this biological clockwork. The cycling of the PER and CRY clock proteins has been thought to be necessary to keep the mammalian clock ticking.
RESULTS - We provide a novel cell-permeant protein approach for manipulating cryptochrome protein levels to evaluate the current transcription and translation feedback model of the circadian clockwork. Cell-permeant cryptochrome proteins appear to be functional on the basis of several criteria, including the abilities to (1) rescue circadian properties in Cry1(-/-)Cry2(-/-) mouse fibroblasts, (2) act as transcriptional repressors, and (3) phase shift the circadian oscillator in Rat-1 fibroblasts. By using cell-permeant cryptochrome proteins, we demonstrate that cycling of CRY1, CRY2, and BMAL1 is not necessary for circadian-clock function in fibroblasts.
CONCLUSIONS - These results are not supportive of the current version of the transcription and translation feedback-loop model of the mammalian clock mechanism, in which cycling of the essential clock proteins CRY1 and CRY2 is thought to be necessary.
For alveolar type I cells, phenotype plasticity and physiology other than gas exchange await further clarification due to in vitro study difficulties in isolating and maintaining type I cells in primary culture. Using an established in vitro model of human fetal type II cells, in which the type II phenotype is induced and maintained by adding hormones, we assessed for transdifferentiation in culture toward a type I-like cell with hormone removal for up to 144 h, followed by electron microscopy, permeability studies, and RNA and protein analysis. Hormone withdrawal resulted in diminished type II cell characteristics, including decreased microvilli, lamellar bodies, and type II cell marker RNA and protein. There was a simultaneous increase in type I characteristics, including increased epithelial cell barrier function indicative of a tight monolayer and increased type I cell marker RNA and protein. Our results indicate that hormone removal from cultured human fetal type II cells results in transdifferentiation toward a type I-like cell. This model will be useful for continued in vitro studies of human fetal alveolar epithelial cell differentiation and phenotype plasticity.
Novel biocompatible macromolecular vectors were developed that not only enable transport of bioactive cargo across the cell membrane but also control the delivery into defined intracellular compartments. This work describes the synthesis and design of two non-peptidic fluorescently labeled Newkome-type dendrimers, differentiated over a varied alkyl spacer with guanidine end moieties. The internalization of the fluorescein-labeled molecular transporter into mammalian cells showed strong subcellular localizations, evident with both live cells and fixed cells costained with DAPI, a nuclear stain. We observed that the subcellular distribution of these vectors varied significantly, as one of the vectors concentrates in the nucleus (FD-1) while the other (FD-2) concentrates in the cytosol. All experiments performed with NIH-3T3 fibroblasts and human microvascular endothelial cells (HMEC) showed similar results. The differential localization patterns of the two molecular transporters can be controlled through the variation of alkyl spacer length at the terminal generation of the dendrimer. Intracellular delivery of bioactive entities into specific subcellular locations, utilizing this practical approach, might overcome limitations in drug delivery and pioneer future technologies in drug transport.
The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.
Bacterial pore-forming toxins have traditionally been thought to function either by causing an essentially unrestricted flux of ions and molecules across a membrane or by effecting the transmembrane transport of an enzymatically active bacterial peptide. However, the Helicobacter pylori pore-forming toxin, VacA, does not appear to function by either of these mechanisms, even though at least some of its effects in cells are dependent on its pore-forming ability. Here we show that the VacA channel exhibits two of the most characteristic electrophysiological properties of a specific family of cellular channels, the ClC channels: an open probability dependent on the molar ratio of permeable ions and single channel events resolvable as two independent, voltage-dependent transitions. The sharing of such peculiar properties by VacA and host ClC channels, together with their similar magnitudes of conductance, ion selectivities, and localization within eukaryotic cells, suggests a novel mechanism of toxin action in which the VacA pore largely mimics the electrophysiological behavior of a host channel, differing only in the membrane potential at which it closes. As a result, VacA can perturb, but not necessarily abolish, the homeostatic ionic imbalance across a membrane and so change cellular physiology without necessarily jeopardizing vitality.
Polyunsaturated fatty acids (PUFAs) and leukotriene B(4) (LTB(4)) are involved in many inflammatory and physiological conditions. The role of arachidonic acid (AA) and linoleic acid (LA) in promoting the assembly of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits is well known, but the involvement of LTB(4) and other 5-lipoxygenase (5-LO) pathway metabolites of AA in hydrogen peroxide (H(2)O(2)) production by PUFA-stimulated polymorphonuclear leukocytes (PMNs) has not been investigated. We examined this question by determining H(2)O(2) production as well as phosphorylation and membrane translocation of the p47phox subunit of NADPH oxidase. Elicited peritoneal PMNs from rats and from 5-LO-deficient or wild-type mice were pretreated with or without inhibitors of LT biosynthesis and antagonists of the receptors for LTB(4) and cysteinyl LTs for 20 min before stimulation with AA (at 5 and 20 microM) or LA (at 20 microM). PUFAs elicited H(2)O(2) production in a dose-dependent manner, and pharmacologic or genetic inhibition of LT synthesis decreased H(2)O(2) production by approximately 40% when compared with untreated controls. LTB(4) was the moiety responsible for H(2)O(2) production, as revealed by studies using receptor antagonists and its exogenous addition. LTB(4) itself also promoted p47phox phosphorylation and translocation. These results identify a heretofore unrecognized role for activation of 5-LO and subsequent production of LTB(4) in stimulation of PMN NADPH oxidase activation by PUFAs.
BACKGROUND - Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells.
RESULTS - In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC) or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH6). The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF)3K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4 degrees C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes), and did not require localization of the enzyme to the nucleus.
CONCLUSIONS - The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells.