Biomedical subjects

K L Cepek

Publications and source records attributed to K L Cepek.

10 recordsLinked to original sources

PubMed1997

Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression.

Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters. To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP. Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h). In vitro transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor. Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes.

DNA-Binding Proteins↗

PubMed1996

Sequence-specific DNA binding of the B-cell-specific coactivator OCA-B.

B-cell-specific transcription of immunoglobulin genes is mediated by the interaction of a POU domain containing transcription factor Oct-1 or Oct-2, with the B-cell-specific coactivator OCA-B (Bob-1, OBF-1) and a prototype octamer element. We find that OCA-B binds DNA directly in the major groove between the two subdomains of the POU domain, requiring both an A at the fifth position of the octamer element and contact with the POU domain. An amino-terminal fragment of OCA-B binds the octamer site in the absence of a POU domain with the same sequence specificity. Coactivator OCA-B may undergo a POU-dependent conformational change that exposes its amino terminus, allowing it to recognize specific DNA sequences in the major groove within the binding site for Oct-1 or Oct-2. The recognition of both the POU domain and the octamer sequence by OCA-B provides a mechanism for differential regulation of octamer sites containing genes by the ubiquitous factor Oct-1.

B-Lymphocytes↗

PubMed1996

Expression of a candidate cadherin in T lymphocytes.

Cadherins are homotypic adhesion molecules that classically mediate interactions between cells of the same type in solid tissues. In addition, E-cadherin is able to support homotypic adhesion of epidermal Langerhans cells to keratinocytes (Tang, A., Amagai, M., Granger, L. G., Stanley, J. R. & Udey, M. C. (1993) Nature (London) 361, 82-85) and heterotypic adhesion of mucosal epithelial cells to E-cadherin-negative intestinal intraepithelial T lymphocytes. Thus, we hypothesized that cadherins may play a wider role in cell-to-cell adhesion events involving T lymphocytes. We searched for a cadherin or cadherins in T lymphocytes with a pan-cadherin antiserum and antisera against alpha- or beta-catenin, molecules known to associate with the cytoplasmic domain of cadherins. The anti-beta-catenin antisera coimmunoprecipitated a radiolabeled species in T-lymphocyte lines that had a molecular mass of 129 kDa and was specifically immunoblotted with the pan-cadherin antiserum. Also, the pan-cadherin antiserum directly immunoprecipitated a 129-kDa radiolabeled species from an 125I surface-labeled Jurkat human T-cell leukemic cell line. After V8 protease digestion, the peptide map of this pan-cadherin-immunoprecipitated, 129-kDa species exactly matched that of the 129-kDa species coimmunoprecipitated with the beta-catenin antiserum. These results demonstrate that T lymphocytes express a catenin-associated protein that appears to be a member of the cadherin superfamily and may contribute to T cell-mediated immune surveillance.

Animals↗

PubMed1994

Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin.

In contrast to sessile cell types, lymphocytes migrate through the vasculature to become diffusely distributed in tissues or organized in lymphoid structures. A complex array of adhesion molecules including selectins, integrins and their counter-receptors mediate lymphocyte homing and migration into tissues and may be constitutively expressed or induced. However, the molecules that mediate the tissue-specific retention of lymphocytes within the parenchyma have not been identified. Along the epithelium at the basolateral surface of enterocytes, intestinal intraepithelial lymphocytes are found. These T cells of the mucosal immune system serve as a model for the tissue-specific compartmentalization of lymphocytes. We investigated whether the localization of these intestinal intraepithelial lymphocytes could be mediated by specific interactions between adhesion molecules expressed selectively on this subpopulation of T cells and tissue-restricted adhesion molecules on epithelial cells. Here we show that heterotypic adhesive interactions between epithelial cells and intraepithelial lymphocytes in vitro are mediated by E-cadherin and the alpha E beta 7 integrin.

Animals↗

PubMed1994

Molecular cloning of the human mucosal lymphocyte integrin alpha E subunit. Unusual structure and restricted RNA distribution.

The human mucosal lymphocyte-1 (HML-1) antigen is expressed on a subclass of T-lymphocytes known as intra-epithelial lymphocytes which are located between mucosal epithelial cells. The HML-1 complex is known to mediate adhesion of intra-epithelial T-lymphocytes to epithelial cell monolayers in vitro. We and others have shown that the HML-1 antigen is an integrin composed of the beta 7 subunit in association with a novel alpha subunit, alpha E. Here we report the cloning of the alpha E cDNA and its primary amino acid sequence. alpha E contained an inserted or I domain and was more homologous to the other I domain containing integrins than to the cleaved group of integrin alpha subunits. However, alpha E contained a unique extra domain of 55 amino acids located just NH2-terminal to the I domain without counterpart in other integrins. This extra domain contained a stretch of 18 consecutive charged residues and included a proteolytic cleavage site. Thus alpha E is the only I domain containing integrin alpha subunit that is also cleaved, and the cleavage site is distinct from that of members of the cleaved group of integrin alpha subunits. These structural features mark alpha E as an unusual member of the integrin family. High levels of alpha E and beta 7 mRNA were restricted to mucosal lymphocytes supporting the hypothesis that alpha E beta 7 plays a role in the localization or site-specific functions of intra-epithelial T-lymphocytes.

Amino Acid Sequence↗

PubMed1994

Distinct structural and functional epitopes of the alpha E beta 7 integrin.

Intestinal intraepithelial lymphocytes (iIEL) are predominantly CD3+, CD8+ T lymphocytes located above or adjacent to the mucosal basement membrane. Although they are positioned to interact with intercellular luminal antigen or with enterocytes, the function of iIEL remains unknown. Most (> 85%) of the iIEL express the alpha E beta 7 integrin which appears to be involved in the adhesion of lymphocytes to epithelial cells. We report the characterization of three monoclonal antibodies (mAb) termed alpha E7-1, alpha E7-2, and alpha E7-3, that react with the alpha E beta 7 integrin recognized by the previously described mAb HML-1 as demonstrated by identical sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility and charge. Flow cytometric analysis of antibody cross-blocking indicated that these mAb recognize distinct epitopes of alpha E beta 7. While all of the mAb were capable of blocking the adhesion of cultured iIEL to a breast epithelial cell line, only HML-1 and alpha E7-1 (which recognize an identical or closely related epitope) were co-stimulatory with suboptimal concentrations of anti-CD3 mAb in inducing proliferation of cultured iIEL. Thus, these mAb appear to recognize functionally distinct epitopes of alpha E beta 7 and will be useful to study relationships between the structure and function of this integrin.

Animals↗

PubMed1994

Evidence for a structural difference in the CD7 polypeptide on human thymocytes and intraepithelial lymphocytes defined by a new monoclonal antibody, 3D9.

Intestinal intraepithelial lymphocytes (IEL) represent a distinct subpopulation of lymphocytes located on or above the basement membrane and adjacent to the basolateral membrane of enterocytes. Thus IEL are strategically positioned to mediate a response to the uptake of foreign antigens or to the alteration of enterocytes by injury or infection. Because of their unique location, we hypothesized that IEL might selectively express specialized cell surface proteins important in their site-specific localization or function. To identify such proteins, we immunized mice with purified human IEL and identified one monoclonal antibody, 3D9, which was found to react with a majority of IEL but with very few lamina propria lymphocytes (LPL) and weakly with most peripheral blood lymphocytes (PBL). Evaluation of this antibody with two-dimensional gels demonstrated that it reacts with CD7, previously known as an early thymocyte differentiation antigen. Interestingly, unlike all other anti-CD7 monoclonal antibodies, 3D9 identified only occasional thymocytes by immunohistochemistry suggesting that CD7 is structurally different on IEL and thymocytes. However, the CD7 polypeptide immunoprecipitated from IEL and thymocytes appeared identical in SDS-PAGE mobility and in charge by two-dimensional gels, despite being recognized differently by monoclonal antibodies. These studies emphasize the expression of CD7 by IEL T cells and reveal the existence of an undefined structural difference between CD7 molecules on IEL compared to thymocytes.

Animals↗

PubMed1994

Reconstitution of cultured intestinal epithelial monolayers with a mucosal-derived T lymphocyte cell line. Modulation of epithelial phenotype dependent on lymphocyte-basolateral membrane apposition.

In vivo, epithelial cells which line the intestine are intimately associated with lymphocytes, termed intraepithelial lymphocytes. Previous studies have demonstrated that intraepithelial lymphocytes are present in the uninflamed mucosa, and become especially prominent in various human enteropathies including coeliac disease, tropical sprue, dermatitis herpetiformis, and giardiasis. Using the intestinal crypt cell line T84, and a previously well-defined human mucosa-derived lymphocyte (MDL) line with phenotypic features similar to (but not specific for) intraepithelial lymphocytes, we describe a co-culture model to study the functional sequellae of MDL-T84 cell interactions in vitro. A co-culture method was defined which permitted reconstitution of the paracellular spaces of physiologically confluent epithelial monolayers with MDL. Such co-cultures thus mimicked the correct geometry of intraepithelial lymphocytes-epithelial cell interactions. The presence of physiologically positioned MDL brought about specific and dramatic effects on intestinal epithelial monolayer function. In a dose-dependent fashion, the presence of MDL significantly attenuated barrier function (expressed as a decrease in monolayer resistance), decreased epithelial electrogenic Cl- secretion, and modulated epithelial-neutrophil interactions. Such effects were not reproduced in monolayers similarly reconstituted with inert polystyrene beads equivalent in size to MDL. These MDL-elicited effects on epithelial function specifically required direct MDL apposition to the epithelial basolateral membrane. Furthermore, this specific form of MDL-epithelial basolateral contact released soluble factors which were able to confer the MDL-reconstituted phenotype on virgin epithelial monolayers in the absence of MDL. We have previously shown that many aspects of the MDL converted epithelial phenotype described here can be induced by IFN-gamma. While IFN-gamma, a cytokine produced by many lymphocytes including intraepithelial lymphocytes, was detectable in conditioned supernatants from co-cultures, it existed at concentrations insufficient to fully explain the physiologic effects observed here.

Cells, Cultured↗

PubMed1993

Integrin alpha E beta 7 mediates adhesion of T lymphocytes to epithelial cells.

Intestinal intraepithelial lymphocytes (iIEL) are a distinct subpopulation of T lymphocytes diffusely distributed in the epithelium. Because > 95% of these iIEL express the integrin alpha E beta 7, we reasoned that this integrin might mediate the localization of iIEL to the epithelium. We report that cultured iIEL bound to mucosal epithelial cells derived from either breast or intestine. This binding was dependent on the presence of divalent cations, and was blocked by mAb specific for two integrins, alpha E beta 7 and alpha L beta 2. After TGF-beta 1 treatment, cultured iIEL cell lines expressed alpha E beta 7 at levels similar to those found on iIEL in vivo. Under these conditions this integrin appeared to predominate in mediating the adhesion of iIEL to epithelial cells. Although alpha L beta 2 blocked the adhesion of iIEL to both endothelial and epithelial cells, alpha E beta 7 blocked binding only to epithelial cells suggesting that the ligand for alpha E beta 7 is not expressed on IL-1 beta-treated cultured endothelial cells. iIEL did not readily bind to the apical surface of confluent polarized epithelial cell monolayers. However, iIEL did bind to these cells when tight junctions were disrupted, a treatment that allows redistribution of proteins compartmentalized to the basolateral surface. This binding was also blocked by mAb to alpha E beta 7. In addition, in vitro coculture of iIEL with polarized epithelial cells resulted in basolateral localization of the iIEL. Thus, it is likely that expression of the alpha E beta 7 ligand is restricted to the basolateral membrane of polarized epithelial cells. Because alpha E beta 7 appears to be a predominant integrin on freshly isolated iIEL, we hypothesized that alpha E beta 7-mediated adhesion may be particularly important in cell-to-cell interactions between T cells and epithelial cells in vivo.

Adult↗

PubMed1992

A family of beta 7 integrins on human mucosal lymphocytes.

The heterodimeric protein complex recognized by the human mucosal lymphocyte 1 (HML-1) monoclonal antibody is expressed on 95% of intraepithelial lymphocytes but on only 1-2% of peripheral blood lymphocytes [Cerf-Bensusson, N., Jarry, A., Brousse, N., Lisowska-Grospierre, B., Guy-Grand, D. & Griscelli, C. (1987) Eur. J. Immunol. 17, 1279-1285]. We purified the smaller HML-1 subunit (105 kDa under nonreducing conditions) from hairy-cell leukemia cells and determined the N-terminal amino acid sequence of this chain. The 17 residues determined were identical to the deduced amino acid sequence encoded by an integrin beta 7 cDNA clone [Yuan, Q., Jiang, W.-M., Krissansen, G.W. & Watson, J.D. (1990) Int. Immunol. 2, 1097-1108]. Biochemical analysis of the larger HML-1 subunit (175 kDa under nonreducing conditions) suggested that it was a distinct member of the cleaved group of integrin alpha chains, which we designated alpha E. The beta 7 chain also was associated with the integrin alpha 4 subunit, suggesting that the HML-1 antigen (alpha E beta 7) and alpha 4 beta 7 constitute a beta 7 integrin family on mucosal lymphocytes. Interestingly, regulation of the expression of the HML-1 antigen was reciprocal to that of lymphocyte function-associated molecule 1 in the presence of transforming growth factor beta 1. We suggest that these beta 7 integrins may play a specific role in mucosal localization or adhesion and that the expression of the HML-1 antigen might be regulated by transforming growth factor beta 1 produced at or near epithelial tissues.

Amino Acid Sequence↗