Although the heteromeric combination of type 1 taste receptors 2 and 3 (T1r2 + T1r3) is well established as the major receptor for sugars and noncaloric sweeteners, there is also evidence of T1r-independent sweet taste in mice, particularly so for sugars. Before the molecular cloning of the T1rs, it had been proposed that sweet taste detection depended on ( a ) activation of sugar-gated cation channels and/or ( b ) sugar binding to G protein-coupled receptors to initiate second-messenger cascades. By either mechanism, sugars would elicit depolarization of sweet-responsive taste cells, which would transmit their signal to gustatory afferents. We examined the nature of T1r-independent sweet taste; our starting point was to determine if taste cells express glucose transporters (GLUTs) and metabolic sensors that serve as sugar sensors in other tissues. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we determined that several GLUTs (GLUT2, GLUT4, GLUT8, and GLUT9), a sodium–glucose cotransporter (SGLT1), and two components of the ATP-gated K + (K ATP ) metabolic sensor [sulfonylurea receptor (SUR) 1 and potassium inwardly rectifying channel (Kir) 6.1] were expressed selectively in taste cells. Consistent with a role in sweet taste, GLUT4, SGLT1, and SUR1 were expressed preferentially in T1r3-positive taste cells. Electrophysiological recording determined that nearly 20% of the total outward current of mouse fungiform taste cells was composed of K ATP channels. Because the overwhelming majority of T1r3-expressing taste cells also express SUR1, and vice versa, it is likely that K ATP channels constitute a major portion of K + channels in the T1r3 subset of taste cells. Taste cell-expressed glucose sensors and K ATP may serve as mediators of the T1r-independent sweet taste of sugars.

apoptosis ͉ mitochondria ͉ Bcl-2 family ͉ tumor suppressor

Interactions between Escherichia coli K1, which causes meningitis in neonates, and macrophages have not been explored well. In this study we found that E. coli K1 was able to enter, survive, and replicate intracellularly in both murine and human macrophage cell lines, as well as in monocytes and macrophages of newborn rats. In addition, we demonstrated that OmpA ؉ E. coli also enters and replicates in human peripheral blood monocytes in vitro. Outer membrane protein A (OmpA) expression on E. coli contributes to binding to macrophages, phagocytosis, and survival within macrophages. Opsonization with either complement proteins or antibody is not required for uptake and survival of the bacteria within the macrophages. Transmission electron microscopy and immunocytochemistry studies with the infected macrophages indicated that OmpA ؉ E. coli multiplies enormously in a single phagosome and bursts the cell. Internalization of OmpA ؉ E. coli by RAW 264.7 cells occurred by both actin-and microtubule-dependent processes, which are independent of RGD-mediated integrin receptors. Internalization and intracellular survival within phagocytic cells thus may play an important role in the development of bacteremia, which is crucial for E. coli crossing of the blood-brain barrier.Escherichia coli is one of the leading gram-negative bacteria that cause neonatal meningitis. The rates of mortality and neurologic sequelae remain high despite advances in antimicrobial therapy (17,19). The two important steps in the pathogenesis of neonatal meningitis are the development of bacteremia with intravascular growth and passage of bacteria across the blood-brain barrier. Studies have shown that a certain threshold of bacteremia is necessary for the development of meningitis in an experimental rat model of hematogenous meningitis (14, 29). Thus, E. coli must avoid host defense mechanisms and proliferate either in the blood or in tissues to maintain a high level of bacteremia, which leads to the onset of meningitis. Invasion and intracellular survival of E. coli, therefore, represent an important pathogenicity mechanism in this infection. Although several E. coli structures and/or genes have been shown to be essential for interaction with brain microvascular endothelial cells (BMEC) (14,30,36,42), a single layer of cells lining the blood-brain barrier, very little is known about the interaction with phagocytic cells.Studies with BMEC have suggested that outer membrane protein A (OmpA) of E. coli plays an important role in the invasion by interacting with a 96-kDa glycoprotein on human BMEC (HBMEC) (25,27). Interaction of OmpA with its receptor induces actin condensation at the E. coli binding site (28). The actin reorganization induced by E. coli depends on activation of several host proteins involved in signaling, including focal adhesion kinase, PI3-kinase, PKC-␣, and caveolin-1 (32,33,37,38). The activated molecules accumulate along with actin during the invasion of HBMEC. In addition, it has recently been shown that OmpA also binds to a c...

Responses in the amiloride-insensitive (AI) pathway, one of the two pathways mediating salty taste in mammals, are modulated by the size of the anion of a salt. This "anion effect" has been hypothesized to result from inhibitory transepithelial potentials (TPs) generated across the lingual epithelium as cations permeate through tight junctions and leave their larger and less permeable anions behind (Ye et al., 1991). We tested directly the necessity of TPs for the anion effect by measuring responses to NaCl and Na-gluconate (small and large anion sodium salts, respectively) in isolated taste cells from mouse circumvallate papillae. Using calcium imaging, we identified AI salt-responsive type III taste cells and demonstrated that they compose a subpopulation of acid-responsive taste cells. Even in the absence of TPs, many (66%) AI salt-responsive type III taste cells still exhibited the anion effect, demonstrating that some component of the transduction machinery for salty taste in type III cells is sensitive to anion size. We hypothesized that osmotic responses could explain why a minority of type III cells (34%) had AI salt responses but lacked anion sensitivity. All AI type III cells had osmotic responses to cellobiose, which were significantly modulated by extracellular sodium concentration, suggesting the presence of a sodium-conducting osmotically sensitive ion channel. However, these responses were significantly larger in AI type III cells that did not exhibit the anion effect. These findings indicate that multiple mechanisms could underlie AI salt responses in type III taste cells, one of which may contribute to the anion effect.

Escherichia coli is one of the most important pathogens involved in the development of neonatal meningitis in many parts of the world. Traversal of E. coli across the blood-brain barrier is a crucial event in the pathogenesis of E. coli meningitis. Our previous studies have shown that outer membrane protein A (OmpA) expression is necessary in E. coli for a mechanism involving actin filaments in its passage through the endothelial cells. Focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) have also been activated in host cells during the process of invasion. In an attempt to elucidate the mechanisms leading to actin filament condensation, we have focused our attention on protein kinase C (PKC), an enzyme central to many signaling events, including actin rearrangement. In the current study, specific PKC inhibitors, bisindolmaleimide and a PKC-inhibitory peptide, inhibited E. coli invasion of human brain microvascular endothelial cells (HBMEC) by more than 75% in a dose-dependent manner, indicating a significant role played by this enzyme in the invasion process. Our results further showed that OmpA؉ E. coli induces significant activation of PKC in HBMEC as measured by the PepTag nonradioactive assay. In addition, we identified that the PKC isoform activated in E. coli invasion is a member of the conventional family of PKC, PKC-␣, which requires calcium for activation. Immunocytochemical studies have indicated that the activated PKC-␣ is associated with actin condensation beneath the bacterial entry site. Overexpression of a dominant negative mutant of PKC-␣ in HBMEC abolished the E. coli invasion without significant changes in FAK phosphorylation or PI3K activity patterns. In contrast, in HBMEC overexpressing the mutant forms of either FAK or PI3K, E. coli-induced PKC activation was significantly blocked. Furthermore, our studies showed that activation of PKC-␣ induces the translocation of myristoylated alanine-rich protein kinase C substrate, an actin cross-linking protein and a substrate for PKC-␣, from the membrane to cytosol. This is the first report of FAK-and PI3K-dependent PKC-␣ activation in bacterial invasion related to cytoskeletal reorganization.

Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5 rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Escherichia coli K1 is the most frequent causative agent of neonatal meningitis. The pathogenic mechanisms of E. coli have been studied by utilizing brain microvascular endothelial cells (BMEC) as an in vitro blood-brain barrier (BBB) model (17,19,20). These studies suggest that S fimbriae mediate attachment to BMEC via NeuAc2,3-Gal epitopes of BMEC surface glycoproteins; however, they do not play a significant role in invasion (25). More-intimate attachment by the bacterial outer membrane protein A (OmpA) mediates the invasion process (15). A similar phenomenon has been identified in the pathogenesis of Neisseria gonorrhoeae, where pili promote initial adherence followed by Opa-mediated interaction for invasion (10). In addition to OmpA, other bacterial factors such as IbeA, IbeB, TraJ, and CNF also play roles in E. coli invasion of BMEC; however, OmpA appears to be the most important factor (2, 6, 7, 9). OmpA ϩ E. coli induces actin rearrangements at the site of bacterial entry, which are completely abolished by treatment of the bacteria with GlcNAc␤1-4GlcNAc polymers, which are receptor analogs (16,17). Computer simulation studies of the interactions between OmpA and GlcNAc␤1-4GlcNAc epitopes indicate that these sugars have more favorable energy than any other sugar molecule tested in our experiments (4). These results are in good agreement with earlier studies in which GlcNAc␤1-4GlcNAc moieties showed significant blocking of E. coli invasion both in vitro and in vivo (16).In support of the role of OmpA in E. coli K1 invasion, studies have also demonstrated that OmpA ϩ E. coli induces the phosphorylation of focal adhesion kinase (FAK) and its interaction with phosphatidylinositol 3-kinase (PI3K) (20, 21). Furthermore, GlcNAc␤1-4GlcNAc polymers blocked the activation of FAK, although at higher concentrations, indicating the role of the human BMEC (HBMEC) receptor for OmpA in transducing signals for internalization of E. coli. In addition, it was shown that E. coli also induces the activation of protein kinase C alpha (PKC-␣) in an OmpA-dependent manner (27). The activated PKC-␣ is recruited to the plasma membrane, where it interacts with caveolin-1, a protein marker for caveolae, for internalization of E. coli (28). Several receptors, such as epidermal growth factor and fibroblast growth factor (14), have been shown to accumulate in caveolae, suggesting that the OmpA receptor could be part of caveolae during...

The multidomain proapoptotic protein Bax of the Bcl-2 family is a central regulator for controlling the release of apoptogenic factors from mitochondria. Recent evidence suggests that the Baxassociating protein MOAP-1 may act as an effector for promoting Bax function in mitochondria. Here, we report that MOAP-1 protein is rapidly up-regulated by multiple apoptotic stimuli in mammalian cells. MOAP-1 is a short-lived protein (t1 ͞2 Ϸ 25 min) that is constitutively degraded by the ubiquitin-proteasome system. Induction of MOAP-1 by apoptotic stimuli ensues through inhibition of its polyubiquitination process. Elevation of MOAP-1 levels sensitizes cells to apoptotic stimuli and promotes recombinant Bax-mediated cytochrome c release from isolated mitochondria. Mitochondria depleted of short-lived proteins by cycloheximide (CHX) become resistant to Bax-mediated cytochrome c release. Remarkably, incubation of these mitochondria with in vitrotranslated MOAP-1 effectively restores the cytochrome c releasing effect of recombinant Bax. We propose that apoptotic stimuli can facilitate the proapoptotic function of Bax in mitochondria through stabilization of MOAP-1.apoptosis ͉ Bcl-2 family ͉ proteasome ͉ DNA damage

Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.