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Biomedical subjects

B Sugden

Publications and source records attributed to B Sugden.

At least 19 recordsLinked to original sources
PubMed2001

CD40 and LMP-1 both signal from lipid rafts but LMP-1 assembles a distinct, more efficient signaling complex.

CD40, a member of the TNFR-1 receptor family, shares several features with LMP-1, an oncoprotein encoded by Epstein-Barr virus. CD40 and LMP-1 activate transcription by binding to TRAFs, JAK3 and/or TRADD. CD40's association with CD40L activates signaling. However, LMP-1 signals independently of a ligand but dependently on self-association. We demonstrate that activated CD40 and LMP-1 co-localize in lipid rafts and recruit TRAF3 there, findings consistent with signals of CD40 and LMP-1 being initiated from lipid rafts. To elucidate their signaling, we compared requirements for their aggregation and subcellular localization. Targeting CD40's monomeric C-terminal signaling domain to lipid rafts activates signaling, as does rendering it trimeric. Addition of both modifications supports signaling more efficiently. Parallel experiments with LMP-1 indicate that targeting the monomeric C-terminal signaling domain of LMP-1 to lipid rafts activates signaling, but trimerizing it does not. Fusing LMP-1's N-terminus and membrane-spanning domains to CD40's C-terminus supports signaling more efficiently than CD40 plus ligand or CD40's trimerized and/or localized derivatives. An activity of LMP-1's N-terminus and membrane-spanning domains other than trimerization must contribute to its efficient signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Importance of cryptic myelin basic protein epitopes in the pathogenicity of acute and recurrent anterior uveitis associated with EAE.

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU), which can relapse without recurring of EAE. In this study, we analyzed the repertoire of MBP epitopes that play a role in acute and recurrent AU by injection of MBP synthetic peptides. In addition to the encephalitogenic epitopes 69-89 and 87-99, several cryptic epitopes were found to be strongly uveitogenic in Lewis rats upon immunization with synthetic peptides, including 100-120, 121-140 and 142-167. However, the peptide corresponding to the MBP residues 1-20 was uniquely capable of inducing AU without EAE. Immunization with intact MBP was not essential for the induction of the recurrence of AU. The responses of T cells from lymph nodes and spleens showed a dominant response to the original disease-induced epitope with responses to secondary epitopes. In conclusion, the analysis of pathogenic determinants important for the induction of uveitis provides further evidence that MBP-specific T cells also contribute to the pathogenesis of anterior uveitis. Moreover, this also suggests that a distinct immunoregulatory mechanism exists in the eye and spinal cord because of the uniqueness of the epitope 1-20 in AU but not EAE, and the capability of MBP-specific T cells of inducing AU without EAE.

Acute Disease↗
PubMed2001

The cis-acting family of repeats can inhibit as well as stimulate establishment of an oriP replicon.

Previously we have shown that the establishment of an oriP replicon is dependent on its epigenetic modification, which occurs in only 1 to 10% of proliferating cells (E. R. Leight and B. Sugden, Mol. Cell. Biol. 21:4149-4161, 2001). To gain insights into the cis-acting requirements for the establishment of oriP replicons, we monitored the replication of oriP plasmid derivatives for several weeks following their introduction into cells. In EBNA-1-positive 143B and H1299 cells, plasmids containing only the region of dyad symmetry (DS) of oriP replicated but were lost more rapidly from cells than were oriP plasmids, demonstrating that the family of repeats (FR) of oriP acts in cis to stimulate replication in these cells. Unexpectedly, we found that the DS plasmid was established efficiently in 293/EBNA-1 cells, being lost at a rate of only 8% per cell generation over 24 days posttransfection. However, plasmids containing the FR in addition to the DS of oriP replicated but were lost at a rate of approximately 30% per cell generation in 293/EBNA-1 cells, indicating that the FR inhibits oriP's establishment in this cell line. FR's enhancement of transcription of a promoter in cis and FR's ability to inhibit replication fork movement do not account solely for oriP's inefficient establishment. In addition, DNA looping between FR and DS neither stimulates nor inhibits replication. Deletion of 11 EBNA-1 binding sites in the FR or replacement of the FR with DS sequences, however, does overcome the inhibitory activity of the FR, thereby allowing efficient establishment of the oriP derivative in 293/EBNA-1 cells.

Binding Sites↗
PubMed2001

Establishment of an oriP replicon is dependent upon an infrequent, epigenetic event.

Plasmids containing oriP, the latent origin of replication for Epstein-Barr virus, support efficient replication in selected cell clones when the viral protein EBNA-1 is provided, being lost at a rate of 2 to 4% per cell generation after removal of selection (A. L. Kirchmaier and B. Sugden, J. Virol. 69:1280-1283, 1995; B. Sugden and N. Warren, Mol. Biol. Med. 5:85-94, 1988). We refer to these plasmids as established replicons in that they support efficient DNA synthesis and partitioning each cell cycle. Unexpectedly, we have found that upon introduction of oriP plasmids into a population of EBNA-1-positive cells, oriP plasmids replicate but are lost precipitously from cells during 2 weeks posttransfection (>25% rate of loss per cell generation). Upon investigation of these disparate observations, we have found that only 1 to 10% of cells transfected with an oriP plasmid expressing EBNA-1 and hygromycin phosphotransferase give rise to drug-resistant clones in which the oriP replicon is established. A hereditable alteration in these drug-resistant cell clones, manifested at the genetic or epigenetic level, does not underlie the establishment of oriP, as newly introduced oriP plasmids replicate but are also lost rapidly from these cells. In addition, a genetic alteration in the oriP plasmid is not responsible for establishment, as oriP plasmids isolated from an established cell clone, propagated in Escherichia coli, and reintroduced into EBNA-1-positive cells are likewise established inefficiently. Our findings demonstrate that oriP replicons are not intrinsically stable in EBNA-1-positive cell lines. Rather, the establishment of an oriP replicon is conferred upon the replicon by a stochastic, epigenetic event that occurs infrequently and, therefore, is detected in only a minority of cells.

Cell Line↗
PubMed2000

Epitope recognition and T cell receptors in recurrent autoimmune anterior uveitis in Lewis rats immunized with myelin basic protein.

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Rats recover and become resistant to further reinduction of EAE. We investigated whether the resistance to reinduction of EAE was associated with the resistance to AU in LEW rats reinjected with MBP. We demonstrated that while rats remained resistant to EAE, they become susceptible to uveitis after recovery, and suffered a second episode of disease. The susceptibility to reinduced disease was associated with the recognition of new MBP epitopes. In contrast to the initial episode of AU, TCR Vbeta8.2 predominance was not observed in the iris/ciliary body. Our results suggest that T cells specific for MBP, which are rapidly reactivated when re-exposed to antigen, are sufficient to induce clinical uveitis in LEW rats. This process may involve a shifting of T cell specificity from the major encephalitogenic peptide utilizing the Vbeta8.2 receptor to a more diverse cell repertoire.

Amino Acid Sequence↗
PubMed2000

The amino-terminus and membrane-spanning domains of LMP-1 inhibit cell proliferation.

The LMP-1 oncoprotein of EBV is required to maintain proliferation of infected B-cells and shares several features with CD40, TNF-R1, and related receptors. Members of this family can bind TRAF and TRADD molecules and activate NF-kappaB and AP-1, as can LMP-1. While CD40 and TNF-R1 are dependent on binding their ligands for their signaling, LMP-1 apparently is not. We have found that LMP-1 can act as a governor of cell proliferation and thereby limit its own activities. Its inhibition of proliferation is not mediated by apoptosis but results in cytostasis in four cell lines tested. The structural moiety of LMP-1 that distinguishes it from CD40 and TNF-R1, its amino-terminus and multiple membrane spanning segments, alone can mediate its cytostatic activity.

Animals↗
PubMed2000

Latent membrane protein 1 of Epstein-Barr virus inhibits as well as stimulates gene expression.

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) functionally resembles a constitutively active, CD40-like receptor and contributes to the maintenance of proliferation of EBV-infected primary human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it binds TRAF, TRADD, and JAK molecules to activate NF-kappaB-, AP-1-, and STAT-dependent pathways as does CD40. Yet LMP-1 appears to lack a ligand to regulate its signaling. We have found that LMP-1, when expressed at physiologic levels, inhibits gene expression detectably. Higher levels of LMP-1 expression eventually inhibit both the steady-state level of RNA produced from a BamHI C promoter reporter and general cellular protein synthesis. These findings indicate that LMP-1 can limit its signaling and that this control is manifest at two levels. The domain of LMP-1 that binds TRAF, TRADD, and JAK/STAT molecules is not required for this regulation. A derivative of LMP-1 that contains only its amino-terminal and membrane-spanning domains is sufficient to inhibit reporter activity when the reporter genes are expressed from the BamHI C and LMP-1 promoters. This same derivative of LMP-1 in parallel assays is sufficient to inhibit wild-type LMP-1's stimulation of NF-kappaB-dependent gene expression. We suggest that LMP-1 encodes stimulatory and inhibitory activities; the latter could limit signaling in the apparent absence of ligand-dependent down-regulation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Using scenarios in chronic disease management guidelines for primary care.

The Prodigy system is a guideline-based decision-support system designed to assist general practitioners in England choose the appropriate therapeutic action for their patients. As part of the system, we developed a novel model for encoding clinical guidelines for managing patients with chronic diseases such as asthma and hypertension. The model structures a guideline as a set of choices to be made by the clinician. It models patient scenarios which drive decision making and are used to synchronize the management of a patient with guideline recommendations. The model is robust with respect to available input data and leaves the control of decision-making to the clinician. We have built execution engines to verify the computability of the model. We intend to test the model integrated in up to 200 live systems from at least four system vendors in English General practice.

Angina Pectoris↗
PubMed1999

The linking regions of EBNA1 are essential for its support of replication and transcription.

The ability of distant cis-acting DNA elements to interact functionally has been proposed to be mediated by the interaction of proteins associated site specifically with those cis-acting elements. We have found that the DNA-linking regions of EBNA1 are essential for its contribution to both replication and transcription. The synthesis of plasmids containing the Epstein-Barr virus (EBV) origin of plasmid replication (oriP) can be mediated entirely by the cellular machinery; however, the replicated molecules are lost rapidly from proliferating cells. When EBNA1 is provided in trans, plasmids containing oriP (oriP plasmids) are synthesized during repeated S phases, and the newly formed daughter molecules are precisely segregated to the daughter cells. The contribution(s) of EBNA1 to the stable replication of oriP plasmids is therefore likely to be postsynthetic. In latently infected cells, EBNA1 also regulates the expression of multiple EBV promoters located as many as 10 kbp away. EBNA1 supports replication and transcription through binding to oriP; both the ability of EBNA1 to bind to DNA and the integrity of its binding sites in oriP are required. However, DNA binding by EBNA1 is not sufficient to support replication or transcription, indicating that an additional activity (or activities) is required. EBNA1 links DNAs to which it binds and can form a loop between the two subelements of oriP, the family of repeats and the region of dyad symmetry, each of which contains multiple binding sites for EBNA1. We have constructed a set of derivatives of EBNA1 which contain both, one, or neither of its linking regions in various contexts. Analyses of these derivatives demonstrate that the linking regions of EBNA1 are essential for its support of replication and transcription and that the ability of derivatives of EBNA1 to link DNAs correlates strongly with their support of these activities in cells. These findings indicate that protein-protein associations of the linking regions of EBNA1 underlie its long-range contributions to replication and transcription.

Cell Line↗
PubMed1999

The PRODIGY project--the iterative development of the release one model.

We summarise the findings of the first two research phases of the PRODIGY project and describe the guidance model for Release One of the ensuing nationally available system. This model was a result of the iterative design process of the PRODIGY research project, which took place between 1995 and 1998 in up to 183 general practices in the England. Release One of PRODIGY is now being rolled out to all (27,000) General Practitioners in England during 1999-2000.

Attitude to Computers↗
PubMed1999

The TextBase project--implementation of a base level message supporting electronic patient record transfer in English general practice.

The TextBase project is a laboratory experiment to assess the feasibility of a common exchange format for sending a transcription of the contents of the Electronic Patient Record (EPR) between different general practices, when patients move from one practice to another in the NHS in England. The project was managed using a partnership arrangement between the four EPR systems vendors who agreed to collaborate and the project team. It lasted one year and consisted of an iterative design process followed by creation of message generation and reading modules within the collaborating EPR systems according to a software requirement specification created by the project team. The paper describes the creation of a common record display format, the implementation of transfer using a floppy disk in the lab, and considers the further barriers before a national implementation might be achieved.

Family Practice↗
PubMed1998

Fusions between Epstein-Barr viral nuclear antigen-1 of Epstein-Barr virus and the large T-antigen of simian virus 40 replicate their cognate origins.

Epstein-Barr viral nuclear antigen-1 (EBNA-1) is required for the stable replication of plasmids that contain oriP, the origin of DNA synthesis used during the latent phase of the Epstein-Barr virus life cycle. EBNA-1 acts post-synthetically through unknown mechanisms to facilitate the continued synthesis of oriP plasmids in ensuing S phases. In contrast to viral replicons such as that of SV40, DNA synthesis of oriP is restricted to a single round during each cell cycle. Large T-antigen of SV40 is a DNA helicase and activates the synthesis of SV40 DNA by recruiting cellular proteins required for DNA synthesis to the origin of SV40. Using fusion proteins of EBNA-1 and large T-antigen, we tested whether tethering large T-antigen to oriP is sufficient to initiate multiple rounds of DNA synthesis from oriP during each cell cycle. We report here that, although these fusion proteins retain the biological activities of both EBNA-1 and large T-antigen, their constituent proteins do not confer the properties of one on the other. Thus, it is not possible to subvert the cellular controls that restrict DNA synthesis from oriP to a single round per cell cycle. These results also provide insights into architectural constraints at oriP and at the SV40 ori.

Antigens, Polyomavirus Transforming↗
PubMed1998

The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.

Bromodeoxyuridine↗
PubMed1998

Antibodies to recoverin induce apoptosis of photoreceptor and bipolar cells in vivo.

Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy (CAR) syndrome. In these studies we examined the effect of anti-recoverin antibodies from the sera of patients with CAR and rat monoclonal antibody on the retinas of Lewis rats. Anti-recoverin autoanti-bodies penetrated into the photoreceptor and bipolar cell layers following intravitreal injection. Their presence in the retina could be detected by immunofluorescence 24 h after injection. At the same time, individual cells undergoing apoptosis were identified throughout photoreceptor and bipolar cell layers using terminal transferase-mediated dUTP nick-end labeling (TUNEL) and electron microscopy. Normal antibodies used in control experiments did not produce TUNEL labeling. At 24 h, DNA fragmentation was confirmed by DNA ladder electrophoresis. At the electron microscopic level, there was clear evidence of cells undergoing apoptotic cell death in the retinas treated with anti-recoverin antibodies. At 24 and 96 h, nuclear chromatin condensation and increased vacuolization of photoreceptor outer segments were observed. An examination of retinas from animals receiving anti-retinal antibodies revealed a loss of 1-2 rows of nuclei in the outer and inner nuclear layers whereas all controls (sham, normal IgG, phosphate buffered saline) showed an unchanged number of nuclei rows. In addition, there was an increase in spacing between the rows of nuclei of the outer nuclear layer in retinas treated with anti- recoverin antibodies, indicating additional cell loss. These studies provide clear evidence that anti-recoverin antibodies are capable of penetrating photoreceptor and bipolar cells, the normal site of recoverin expression in the retina, and that anti-recoverin antibodies produce apoptotic cell death. A similar mechanism may occur in patients with CAR, which may lead to visual loss and blindness.

Animals↗
PubMed1998

Rep*: a viral element that can partially replace the origin of plasmid DNA synthesis of Epstein-Barr virus.

Replication of the Epstein-Barr viral (EBV) genome occurs once per cell cycle during latent infection. Similarly, plasmids containing EBV's plasmid origin of replication, oriP, are replicated once per cell cycle. Replication from oriP requires EBV nuclear antigen 1 (EBNA-1) in trans; however, its contributions to this replication are unknown. oriP contains 24 EBNA-1 binding sites; 20 are located within the family of repeats, and 4 are found within the dyad symmetry element. The site of initiation of DNA replication within oriP is at or near the dyad symmetry element. We have identified a plasmid that contains the family of repeats but lacks the dyad symmetry element whose replication can be detected for a limited number of cell cycles. The detection of short-term replication of this plasmid requires EBNA-1 and can be inhibited by a dominant-negative inhibitor of EBNA-1. We have identified two regions within this plasmid which can independently contribute to this replication in the absence of the dyad symmetry element of oriP. One region contains native EBV sequences within the BamHI C fragment of the B95-8 genome of EBV; the other contains sequences within the simian virus 40 genome. We have mapped the region contributing to replication within the EBV sequences to a 298-bp fragment, Rep*. Plasmids which contain three copies of Rep* plus the family of repeats support replication more efficiently than those with one copy, consistent with a stochastic model for the initiation of DNA synthesis. Plasmids with three copies of Rep* also support long-term replication in the presence of EBNA-1. These observations together indicate that the latent origin of replication of EBV is more complex than formerly appreciated; it is a multicomponent origin of which the dyad symmetry element is one efficient component. The experimental approach described here could be used to identify eukaryotic sequences which mediate DNA synthesis, albeit inefficiently.

Cell Line↗
PubMed1997

Studies on the mechanism of DNA linking by Epstein-Barr virus nuclear antigen 1.

Epstein-Barr virus nuclear antigen 1 (EBNA1) can both bind to and link DNA. Dimers of EBNA1 bind specific sites, two clusters of which, the FR and DS, comprise the necessary cis-acting elements of the Epstein-Barr viral origin of plasmid replication. EBNA1-dimers can link FR and DS, looping out the intervening DNA. EBNA1 can also intermolecularly link DNAs to which it binds. Residues of EBNA1 that can mediate linking have been mapped to at least three, non-overlapping domains. These domains, when fused to the dimerization and DNA-binding domain of GAL4, can self-associate and thereby link DNAs bound site specifically by GAL4. Two disparate mechanisms could underlie self-association of linking domains: 1) linking domains could associate with other linking domains directly, or 2) linking domains could associate indirectly by binding to a common nucleic acid intermediate. We have found that EBNA1 can link DNA by each of these mechanisms, however, the linking domains associate directly with a greater apparent affinity than through a nonspecific nucleic acid intermediate.

Binding Sites↗