Expression of the human CD8a gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis. As an initial step towards understanding the molecular basis for tissue-specific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites, three of which were T cell restricted. By using a reporter-based expression approach, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8a gene. Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element. DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH proteins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat which contained the GATA-3, bHLH, and LyF-1 binding sites. Site-directed mutation of the Ets-1 and GATA-3 sites dramatically reduced enhancer activity. The functional importance of the other binding sites only became apparent when combinations of mutations were analyzed. Taken together, these results suggest that the human CD8ae gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene.Comparison of the CD8ax enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes.The T-lymphocyte differentiation marker CD8 identifies a distinct T-cell subset which can be triggered to express cytotoxic and/or suppressor activity upon recognition of antigenic peptides presented in the context of class I major histocompatibility complex (MHC) molecules (46,62,75). Expressed on the T-cell surface as either an a/ot homodimer or a/1 heterodimer, CD8 is capable of direct extracellular interaction with the a3 domain of MHC class I molecules (12,60,63,68) and intracellular association with the tyrosine kinase p56lck (3,8), assisting both the T-cell receptor (TCR) in antigen recognition and the CD3 complex in signal transduction. Thus, CD8 serves as an integral part of a complex recognition/response apparatus which functions to initiate antigen-driven activation of mature effector T cells in the periphery as well as differentiation of immature T cells in the thymus.Tissue-specific expression of CD8 is under precise developmental control. When bone marrow-derived precursor cells begin the process of thymopoiesis, a panel of T-cellspecific genes (TCR, CD2, CD3, and CD4) including CD8a and CD83 are coordinately expressed in a defined temporal sequence (10,32,45
The CD8 dimer interacts with the a3 domain of major histocompatibility complex class I molecules through two immunoglobulin variable-like domains. In this study a crystal structure-informed mutational analysis has been performed to identify amino acids in the CD8a/a homodimer that are likely to be involved in binding to Recently the crystal structure of a soluble form of the human CD8a/a homodimer was solved (12). In the N-terminal domain of each polypeptide, two sheets consisting of five and four antiparallel (3-strands form a typical immunoglobulin fold. Connecting these strands is a conserved array of loops, three of which are analogous to the complementarity-determining regions of immunoglobulin (CDR1, CDR2, CDR3). Modeling of the electrostatic surface potential of the CD8a/a homodimer has revealed that the membrane-distal, CDR-containing surface of the molecule is predominantly positively charged.The binding site on class I molecules for CD8a/a has been studied in detail. Previous mutational analyses have identifled three clusters of amino acids in the a3 domain that are important for CD8a/a-class I binding and pinpointed one cluster, residues 223-229, as critical for this interaction (1,(13)(14)(15). This cluster is conserved between mouse and human and comprises a highly exposed, negatively charged loop between strands C and D in the a3 domain. In contrast to the body of work that has contributed to the localization of the putative class I binding site for CD8, only one study has focused on defining the complementary interaction site on CD8a/a. Based on the finding that murine CD8a/a did not bind to human HLA class I in a cell-cell adhesion assay, Sanders et al. (16) performed homolog scanning mutagenesis by exchanging human amino acids with their nonconserved murine counterparts. The results ofthis work indicated a role for both the CDR1-like and CDR2-like loops in this speciesspecific interaction. However, since this study focused on the differences between two species, conserved amino acids involved in binding would not have been identified. In addition, the individual amino acids which might be potential contact points could not be determined because these mutants often contained more than one amino acid substitution.In the current study the site of interaction on CD8a/a has been further characterized by using a panel of point mutants generated with the aid of the CD8a/a crystal structure. Analysis of these mutants in a transient cell-cell adhesion assay has identified a core of amino acids that are likely candidates for interaction with class I molecules. Overall, the results support a bivalent model of interaction between one CD8 homodimer and two class I molecules. The role of electrostatic interactions between the predominantly positively charged CD8a/a and the predominantly negatively charged class I a3 domain is also supported by these findings. MATERIALS AND METHODSMutagenesis. Site-directed mutagenesis of the human CD8a cDNA in pBluescript SK(+) was as described (16). To facilitate t...
Emerging Infectious Diseases (bla PER-1 and bla VIM-2) had been detected separately among clinical isolates, underscores the possibility of the emergence of new threatening combinations of resistance determinants among nosocomial pathogens. In fact, the recruitment of similar resistance determinants within a single P. aeruginosa strain can determine a resistance phenotype to virtually all the available antipseudomonal beta-lactams, an occurrence that can be particularly dramatic when, as in the present case, resistance to beta-lactams is associated with resistance against aminoglycosides and fluoroquinolones. In this case, only piperacillin (which appears to be a relatively poor substrate for both enzymes [3,5]) retained moderate activity in vitro and, administered at high dosage in combination with tazobactam, was apparently effective in vivo. Should a similar resistance phenotype disseminate, it might have strategic implications for the development of new betalactamase inhibitors and for selection of beta-lactam compounds to associate wth inhibitors.
(bla PER-1 and bla VIM-2 ) had been detected separately among clinical isolates, underscores the possibility of the emergence of new threatening combinations of resistance determinants among nosocomial pathogens. In fact, the recruitment of similar resistance determinants within a single P. aeruginosa strain can determine a resistance phenotype to virtually all the available antipseudomonal beta-lactams, an occurrence that can be particularly dramatic when, as in the present case, resistance to beta-lactams is associated with resistance against aminoglycosides and fluoroquinolones. In this case, only piperacillin (which appears to be a relatively poor substrate for both enzymes [3,5]) retained moderate activity in vitro and, administered at high dosage in combination with tazobactam, was apparently effective in vivo. Should a similar resistance phenotype disseminate, it might have strategic implications for the development of new betalactamase inhibitors and for selection of beta-lactam compounds to associate wth inhibitors. AcknowledgmentsThis work was supported in part by grant no. FMRX-CT98-0232 from the European Training and Mobility of Researchers Network on metallo-beta-lactamases. Jean-Denis Jamestown Canyon Virus: Seroprevalence in ConnecticutTo the Editor: Jamestown Canyon virus (JCV), a member of the California serogroup, has a wide geographic distribution throughout much of temperate North America. It causes mild febrile illness and, rarely, aseptic meningitis or primary encephalitis (1). JCV has been isolated from mosquitoes each year that surveys have been done in Connecticut, and 28 positive pools from 10 mosquito species were found during 2000 (T. Andreadis, pers. commun.). In contrast, only 14 positive mosquito pools were found to contain West Nile virus (WNV), which has recently been introduced into Connecticut (2). JCV has been isolated from Aedes mosquitoes in Connecticut, and serologic evidence suggests it is widespread in deer (3,4). No recent seroprevalence surveys have been done in Connecticut, nor have any human cases of infection or disease due to JCV been documented. We report the results of two seroprevalence surveys done with standard indirect fluorescent assays (IFA) to detect immunoglobulin G antibodies to JCV. One survey examined 1,086 sera collected in 1990 from blood donors. The second survey examined 1,016 sera submitted to the Connecticut State Public Health Laboratory in 1995.The IFA used JCV-infected baby hamster kidney cells (BHK-21). Infected and uninfected cell suspensions were air dried and fixed onto Teflon-coated, 12-well slides. Prepared slides were stored at -70°C. Sera were tested at a minimum dilution of 1:16. After incubation and washing of the fluorescein-conjugated counterstain, slides were dried and examined by fluorescent microscope (American Optical, Buffalo, NY). The positive human control serum was designated as the 4+ baseline with which the test sera were compared. Selected sera were tested by a serum dilution plaque reduction neutralization test (PRNT) a...
Expression of the human CD8 alpha gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis. As an initial step towards understanding the molecular basis for tissue-specific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites, three of which were T cell restricted. By using a reporter-based expression approach, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8 alpha gene. Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element. DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH proteins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat which contained the GATA-3, bHLH, and LyF-1 binding sites. Site-directed mutation of the Ets-1 and GATA-3 sites dramatically reduced enhancer activity. The functional importance of the other binding sites only became apparent when combinations of mutations were analyzed. Taken together, these results suggest that the human CD8 alpha gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene. Comparison of the CD8 alpha enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes.
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