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U Blank

Publications and source records attributed to U Blank.

At least 37 records · Page 2Linked to original sources
PubMed1997

Effects of nicotine on human nasal epithelium: evidence for nicotinic receptors in non-excitable cells.

We investigated the effects of nicotine and its derivate nicotine di-d-tartrate on primary cultured human nasal epithelial cells. Both substances evoked an increase in the intracellular free calcium concentration. In the presence of extracellular Ca2+ the cytosolic Ca2+ ([Ca2+]i) increase was long lasting, whereas in the absence of external Ca2+ there was a transient increase of [Ca2+]i indicating that nicotine has an influence on Ca2+ conductances across the membranes and on intracellular Ca2+ stores. Both effects could be blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). Apical or basolateral application of nicotine during transepithelial transport measurements with confluent monolayers of cultured human nasal cells resulted in a significant, reversible decrease of amiloride-sensitive sodium absorption with an apparent half-maximal blocker concentration of about 950 microM. To exclude the possibility that remnant neuronal components were responsible for the observed effects we used tetrodotoxin and verapamil to block putative neuronal channels and 4-(4-diethylamino)styryl-N-methylpyridinium iodide (4-di-2-Asp) to stain neuronal tissue. Both experimental approaches demonstrated that there were no neuronal-mediated effects. These results indicate the direct effects of nicotine on human nasal epithelium, giving the first evidence of the existence of nicotinic receptors in non-excitable cells.

Absorption↗
PubMed1996

[Benzamil and mucoviscidosis. Primary culture of nasal mucosa as an electrophysiologic in vitro model].

The genetic disease cystic fibrosis (CF) is characterized by a defect in the gene encoding for an epithelial chloride channel. This gene product, cystic fibrosis transmembrane conductance regulator (CFTR), in turn leads to a decreased chloride secretion. When this occurs sodium absorption is increased drastically because of an up-regulation of the epithelial sodium channel. In airway epithelia these changes in ion permeabilities result in dehydration of the mucus blanket and impaired mucociliary clearance, and subsequently lead to chronic infections of the airways. In this study we established a primary cell culture of human nasal epithelium from CF and non-CF patients. The increased transepithelial potential in CF exclusively was due to the greater sodium absorption. In previous studies, amiloride was found to specifically block the epithelial sodium channel. Inhalation of nebulized amiloride has been used clinically in the treatment of CF to inhibit sodium absorption and exert a secondary effect on water withdrawal. In the present cell culture model benzamil, an amiloride analogue, was found to inhibit sodium absorption completely, but at strikingly lower concentrations than amiloride. This raises the possibility of using benzamil for inhalation and to provide better efficacy in the symptomatic therapy of patients with CF.

Amiloride↗
PubMed1995

Strong similarities in antigen fine specificity among DRB1* 1302-restricted tetanus toxin tt830-843-specific TCRs in spite of highly heterogeneous CDR3.

We investigated the Ag fine specificity of four TCRs that shared the same V beta segment but used V alpha s of three different subfamilies and displayed highly heterogeneous alpha and beta CDR3. The TCRs recognized the tetanus toxin tt830-843 (QYIKANSKFIGITE) epitope presented by DRB1*1302. By using a large panel of monosubstituted peptide analogues, we first defined the requirements for tt830-843 binding to DRB1*1302. We found that three residues, I832, N835, and G840, were critical for the interaction with DRB1*1302. Residues potentially contacted by the four TCRs were functionally defined by measuring the IL-2 response to the analogues. Except for the first and the last three residues, as well as I832 and G340, all of the others appeared to provide contacts with the four TCRs, indicating a considerable overlapping in the way these TCRs interact with the peptide. More importantly, and contrary to expectations, the two TCRs expressing the same V alpha/V beta germ-line segments showed a strikingly similar reactivity toward nearly all substitutions; moreover, more pronounced differences were observed when comparing TCRs using different V alpha segments. These results indicate that TCRs with entirely distinct CDR3s in the context of conserved V segments may not differ substantially in the way they recognize the ligand, and may provide new insights into understanding the formation of TCR/peptide/MHC ternary complexes. During these studies, we noticed that analogues with nonconservative substitutions at I832, which bound very unstably to DRB1*1302, could effectively stimulate T cells, suggesting a role of the TCR in contributing toward stabilization of peptide binding.

Amino Acid Sequence↗
PubMed1995

Regulation of electrogenic Na+ transport across leech skin.

The dorsal integument of the medical leech Hirudo medicinalis exhibits a marked amiloride-sensitive Na+ absorption. With 20 mM Na+ in the apical solution, the transepithelial short-circuit current (Isc) was approximately 40% higher than with 115 mM Na+, whereas the transepithelial potential (VT) with 20 mM Na+ was -35.7 +/- 4.5 and -20.6 +/- 2.6 mV with 115 mM Na+. Amiloride (100 microM) inhibition at 20 mM apical Na+ was also significantly larger than with 115 mM Na+ in the solution. Benzamil (100 microM) showed additional inhibition after amiloride. Large transient overshooting currents occurred only when 115 mM Na+ was added after some minutes of Na(+)-free apical solution. Addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the serosal side in the presence of 115 mM apical Na+ nearly doubled Isc. This cAMP effect was reduced to only 20% in the presence of 20 mM Na+. Guanosine 3',5'-cyclic monophosphate (cGMP) slightly increased Isc, whereas ATP showed biphasic potency. Removal of calcium from the apical side resulted in a large stimulation of amiloride-sensitive Isc only in the presence of 115 mM Na+. When currents were activated with cAMP, a deprivation of Ca2+ modestly reduced the amiloride-sensitive Isc. The Na+ channel of leech integument was found highly selective for Na+ over other monovalent cations. The permeability ratio for Na+ over K+ was approximately 30:1; the selectivity relationship for the investigated cations was Na+ > Li+ > NH4+ > K+ approximately Cs+ approximately Rb+.

Amiloride↗
PubMed1993

Analysis of tetanus toxin peptide/DR recognition by human T cell receptors reconstituted into a murine T cell hybridoma.

We have previously reported that human T cell receptors (TcR) selected in the class II-restricted (HLA-DRB1*1302) response to a tetanus toxin peptide (tt830-843) frequently used the V beta 2 germ-line segment which paired with several V alpha segments and that the putative CDR3 of both alpha and beta chains showed remarkable heterogeneity. To analyze the structural basis for recognition of the tt830-843/DR complex, five of these TcR were reconstituted into a murine T cell hybridoma, 58 alpha- beta-, by expressing the human alpha and beta variable regions joined to the mouse alpha and beta constant regions, respectively. The chimeric TcR, expressing the same V beta germ-line segment (V beta 2), two expressing V alpha 21.1, two V alpha 17.1 and one V alpha 8.1 were shown to have the expected antigen specificity and DR restriction. Two lines of evidence suggested that the putative CDR3, although not conserved in these TcR, played a key role in recognition. First, two TcR with identical V germ-line segments but distinct CDR3 showed large difference in their capacity to react with the ligand. Second, interchanging the alpha and beta chains from tt830-843/DR1302-specific TcR which differed in their CDR3 sequences invariably led to loss of recognition. We also asked whether germ-line V alpha 17.1 could functionally replace V alpha 21.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, V alpha replacement abrogated TcR reactivity. Taken together, these data underline the fine interdependence of the structural components of the TcR binding site in defining a given specificity. Four of the TcR studied displaying promiscuous recognition were also tested against different DR alleles and site-directed mutants. The results of these experiments suggested that, in spite of their structural heterogeneity, anti-tt830-843 TcR may have a similar orientation with respect to the peptide/DR complex. The reconstitution system described herein should represent a valuable tool for detailed studies of human TcR specificity.

Amino Acid Sequence↗
PubMed1993

Expression of the alpha chain of human Fc epsilon RI in transfected rat basophilic leukemia cells: functional activation after sensitization with human mite-specific IgE.

The actual dilemma in studying the binding and triggering capacity of IgE from allergic patients is the lack of cultured basophils or mast cell analogs of human origin. Human IgE binds with exquisite species specificity to the high affinity IgE receptor (Fc epsilon RI) expressed on the surface of these cells. In rodents this receptor has been characterized as a tetrameric plasma membrane protein composed of an IgE-binding alpha chain, a beta chain and two disulfide-linked gamma chains. In order to establish a cell line expressing the alpha chain of human Fc epsilon RI which can be triggered with IgE from human patients and specific allergen, we transfected the cDNA coding for the human alpha subunit into rat basophilic leukemia cells. The resulting transfectants express the human alpha chain on the cell surface in the form of a hybrid complex associated with endogenous rat gamma chains. After sensitization with human IgE from mite-specific patients, the transfectant produces a calcium response upon incubation with allergen. The established cell line can be used as a model system to study the mechanism of mast cell triggering through IgE from allergic patients.

Allergens↗
PubMed1991

Characterization of truncated alpha chain products from human, rat, and mouse high affinity receptor for immunoglobulin E.

The high affinity receptor for immunoglobulin E (IgE) is a tetrameric structure (alpha beta gamma 2) consisting of non-covalently associated subunits: one IgE-binding alpha chain, one 4-fold membrane spanning beta chain, and two disulfide-linked gamma chains. Here, we have engineered alpha cDNA constructs (alpha trunc) encoding exclusively the leader peptide and the extracellular domain of the alpha subunit. Transfection of human alpha trunc into COS-7 cells resulted in the secretion of soluble IgE-binding polypeptides. By contrast, the polypeptides generated from rat and mouse alpha trunc transfections were sequestered in the endoplasmic reticulum and degraded even though they appeared to fold properly as judged by their capacity to bind IgE. Stable transfectants with human alpha trunc were obtained from a dihydrofolate reductase-deficient Chinese hamster ovary cell line. Several clones secreted substantial amounts (0.1 microgram/ml/10(6) cells) of IgE-binding polypeptides. The dissociation rate of bound IgE from this soluble truncated alpha (kappa-1 = 4.9 x 10(-6) s-1 at 25 degrees C) was characteristic of receptors on intact cells. After treatment with tunicamycin, the transfectants secreted unglycosylated 18-kDa polypeptides which could also bind IgE. These unglycosylated products had a tendency to form dimers and higher oligomers which were resistant to treatment by sodium dodecyl sulfate and reducing agents. These data demonstrate unequivocally that the extracellular domain of the alpha subunit is sufficient to mediate high affinity binding of IgE. Furthermore, posttranslational addition of carbohydrates is not required for proper folding and function of the receptor binding site. The truncated human alpha should be a suitable reagent for crystallographic analysis and for detailed analysis of the receptor binding sites.

Animals↗
PubMed1991

The high-affinity receptor for immunoglobulin E: a target for therapy of allergic diseases.

The high-affinity receptor for IgE (Fc epsilon RI) on mast cells and basophils is a tetrameric complex, alpha beta gamma 2. Here we summarize the latest developments on the structure and function of this receptor. By genetic transfer, we have engineered a cell line secreting substantial amounts of a peptide containing exclusively the extracellular domain of the alpha-subunit. This domain by itself is sufficient to mediate high-affinity binding of IgE. Glycosylation and the presence of the other subunits are not necessary for the binding function. The gamma-subunit of Fc epsilon RI is part of other receptors such as Fc gamma RIII and the T cell receptor, and therefore is likely to play an important although still undefined functional role. A detailed knowledge of how the receptor interacts with IgE and induces cellular degranulation may lead to the design of new therapeutic approaches to allergic diseases. The potential strategies are discussed.

Animals↗
PubMed1989

A macrophage Fc gamma receptor and the mast cell receptor for IgE share an identical subunit.

Fc receptors for immunoglobulins are found on many immune cells and trigger essential functions of the immune defence system. With the exception of the high-affinity receptor for immunoglobulin E (Fc epsilon RI), these receptors were thought to consist of single polypeptides. Fc epsilon RI is a tetrameric complex of one alpha-subunit, one beta-subunit and two gamma-subunits. Here we report the cloning of a polypeptide identical to the gamma-chains of Fc epsilon RI, from mouse macrophages that do not express this receptor. Biosynthetic labelling and gene transfer together show that these gamma-chains associate with one of the macrophage receptors (Fc gamma RIIa). The human homologue, Fc gamma RIII (CD16), from natural killer cells is also expected to associate with gamma-chains. It is possible that these gamma-chains and the homologous zeta-chains of the T-cell antigen receptor belong to a new family of related proteins which share a common role in the signal transducing pathway.

Animals↗
PubMed1989

Expression of high-affinity binding of human immunoglobulin E by transfected cells.

The receptor with high affinity for immunoglobulin E (IgE) on mast cells and basophils is critical in initiating allergic reactions. It is composed of an IgE-binding alpha subunit, a beta subunit, and two gamma subunits. The human alpha subunit was expressed on transfected cells in the presence of rat beta and gamma subunits or in the presence of the gamma subunit alone. The IgE binding properties of the expressed human alpha were characteristic of receptors on normal human cells. These results now permit a systematic analysis of human IgE binding and a search for therapeutically useful inhibitors of that binding.

Animals↗
PubMed1989

Complete structure and expression in transfected cells of high affinity IgE receptor.

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, is found exclusively on mast cells and basophils. When multivalent allergens bind to the receptor-bound IgE, the consequent aggregation of the receptors leads to the release of mediators responsible for allergic symptoms. In rodents Fc epsilon RI is a tetrameric complex of non-covalently attached subunits: one IgE-binding alpha subunit, one beta subunit and a dimer of disulphide-linked gamma subunits. Complementary DNA encoding the alpha and the beta subunits has recently been isolated, but expression of IgE-binding by transfected cells has not yet been achieved. Here we report the cloning of cDNA for the gamma subunit, and propose a model for the alpha beta gamma 2 tetramer which accounts for many of the structural features of the receptor. The rodent receptor on the surface of COS 7 cells was expressed only when the cDNAs for all three subunits were cotransfected. Successful expression of human IgE receptors should now be possible, eventually to permit the detailed analysis of the human IgE-receptor interaction and assist the search for therapeutically effective inhibitors.

Amino Acid Sequence↗
PubMed1989

Identification of the Fc gamma RII-related component of murine IgG-BF.

Suppressor murine IgG-BF produced by the T cell hybrid (T2D4) expressing low affinity Fc gamma R (Fc gamma RII) contain four biologically active polypeptides of pI 5.2, 6.3, 7.7 and 8.3, respectively. They were fractionated by affinity chromatography on immunoadsorbents coupled with F(ab')2 fragments of the monoclonal anti-Fc gamma RII antibody 2.4G2 and by hydrophobic interaction chromatography. Both methods led to the identification of biologically active IgG-BF which react with 2.4G2 and of IgG-BF which do not react with 2.4G2. Molecules bearing the epitope recognized by 2.4G2 had an apparent pI of 5.3 while the pI of those which did not express this epitope were 6.3, 7.8 and 8.5, respectively. Therefore, one IgG-BF polypeptide of pI 5.3 is probably related to Fc gamma RII.

Animals↗
PubMed1989

The receptor with high affinity for IgE.

The cDNAs for each of the three types of polypeptide that form the high affinity IgE receptor have been cloned and sequenced. Analysis of the predicted amino acid sequence and other data suggests that the four-chained structure (alpha beta gamma 2) contains seven transmembrane segments. The alpha chain resembles the immunoglobulin-binding chain found in other Fc receptors, but the beta and gamma chain sequences do not resemble other known proteins. (The one exception: the transmembrane segment of the gamma chains, which is homologous to the corresponding segment of the zeta chain of the CD3 complex found on T lymphocytes). Efficient expression of IgE binding by the rat receptor in COS cells was observed only when the coding sequences for each of the three chains were co-transfected. So far, only the cDNA for the human alpha chain has been successfully cloned. We attempted to express this chain by co-transfecting its cDNA with those for the rat beta and gamma chains. Surprisingly, co-transfection with the cDNA for the gamma chain was sufficient, although when the beta and gamma chains were both co-transfected, expression of alpha beta gamma oligomers was evident. Approaches being used to define by genetic manipulation the functional role of various parts of the receptor are discussed.

Animals↗
PubMed1989

Emerging picture of the receptor with high affinity for IgE.

The cDNAs for two of the subunits of the receptor with high affinity for IgE have been isolated and sequenced. That for the IgE-binding alpha subunit predicts a polypeptide with a single transmembrane segment. The predicted extracellular amino terminal portion, comprised of 180 residues, is homologous to the corresponding region of Fc gamma receptors and contains two truncated immunoglobulin-like domains. The cDNA for beta predicts a peptide with four transmembrane segments, but its sequence is unrelated to known proteins. Experiments to isolate the cDNA for the gamma subunit have begun; when completed, cotransfection of the three genes will be attempted to get expression, a process that has not so far been successful for the individual subunits. Such expression will permit further analysis of the receptor's function and ultimately may provide information on how best to intervene clinically in the activation of mast cells and basophils.

Animals↗
PubMed1988

Isolation and characterization of cDNAs coding for the beta subunit of the high-affinity receptor for immunoglobulin E.

Among receptors that bind the Fc region of immunoglobulins ("Fc receptors"), only the one with high affinity for immunoglobulin E (IgE) is known to consist of more than a single polypeptide. In addition to the IgE-binding alpha chain, the receptor contains a single beta chain and two, disulfide-linked, gamma chains. From a cDNA library of a rat mucosal mast cell tumor, from which we recently cloned cDNAs coding for the alpha chain, we have now isolated cDNAs coding for the beta subunit. In vitro transcription-translation of the cDNA directed the synthesis of a polypeptide reactive with two distinctive anti-beta monoclonal antibodies and whose molecular weight was identical to that of authentic beta chains. Polyclonal antibodies to beta peptides expressed in Escherichia coli reacted with intact receptors and isolated beta chains. The gene encodes a protein of 243 residues with no leader sequence. A hydropathicity plot suggests that the polypeptide crosses the plasma membrane four times. The epitope recognized by one of the monoclonal antibodies was localized to the NH2 terminus; that by the other was localized to the COOH terminus. Since those antibodies react with membranes and not with intact cells, we suggest that both ends of the beta subunit are cytoplasmic. RNA transfer blots at high stringency failed to reveal mRNA for beta chains in a variety of cells (in particular, monocytes) that do not contain the high-affinity receptor for IgE.

Amino Acid Sequence↗
PubMed1987

The carbohydrate moieties of suppressor IgG-binding factor released by murine T cells.

The carbohydrate moieties of murine IgG-binding factor (IgG-BF) were studied using lectins binding N-glycosylated sequences such as Concanavalin A (Con A), Lens culinaris agglutinin (LcA), and wheat germ agglutinin (WGA), and lectins binding O-glycosylated sequences such as peanut agglutinin (PNA) and Helix pomatia Agglutinin (HpA). Sources of IgG-BF were: (1) supernatants from T2D4, a T cell hybridoma constitutively producing IgG-BF, and (2) factor purified by affinity chromatography on rabbit IgG-Sepharose, using T2D4 supernatants or supernatants of alloantigen-activated T cells (ATC) as starting material. The presence of IgG-BF was assessed by its ability to inhibit secondary anti-sheep red blood cell (SRBC) IgG antibody responses in vitro and to inhibit rosette formation between Fc gamma receptor (Fc gamma R)-positive spleen cells and erythrocytes sensitized with rabbit anti-Forssman IgG antibodies. Fractionation on immobilized lectins showed that IgG-BF: (1) is completely adsorbed by WGA and PNA and partially by Con A, LcA and HpA, and (2) can be eluted from the five different lectins using the competitor sugars. When produced in the presence of tunicamycin, an inhibitor of N-glycosylation, IgG-BF still binds to HpA which has affinity for O-glycosylated carbohydrate chains. These results indicate that IgG-BF is a glycoprotein with N- and O-glycosylated carbohydrate moieties.

Animals↗