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Biomedical subjects

Edward A Burton

Publications and source records attributed to Edward A Burton.

9 recordsLinked to original sources

Zebrafish DJ-1 is evolutionarily conserved and expressed in dopaminergic neurons.

Loss-of-function mutations in the human PARK7 gene, encoding DJ-1, are a rare cause of autosomal recessive Parkinson's disease (ARPD). To facilitate generation of a novel vertebrate model, in which to examine the biochemical functions of DJ-1 in vivo, we cloned and characterized the zebrafish orthologue of DJ-1 (zDJ-1). The 0.95 kb zDJ-1 mRNA is expressed in adult zebrafish brain, muscle and gut, and in the embryo from 24 h post-fertilization. The zDJ-1 transcript encodes a 19.8 kDa, 189 amino acid protein, which is 83% identical to human DJ-1. Residues thought to be functionally important sites of post-translational modification in human DJ-1, and critical positions affected by pathogenic missense mutations in ARPD patients, are conserved in zDJ-1. The 14 kb zDJ-1 gene contains six exons and is located on zebrafish chromosome 8; the structure of the gene is highly homologous to human DJ-1, except that there are no alternatively spliced non-coding 5' exons. The single zDJ-1 first exon shows 5' end heterogeneity, reflecting multiple transcription start sites. In the adult zebrafish brain, zDJ-1 immunoreactivity was prominent in the cytoplasm of most neurons, and in the neuropil, but was less evident within white matter tracts, consistent with neuronal somatic and dendritic localization. Dopaminergic neurons in each of the major forebrain and diencephalic TH-positive cell groups expressed zDJ-1. These studies show that zDJ-1 is very similar to human DJ-1 and delineate essential resources, allowing further examination of the function and regulation of DJ-1, using the zebrafish as a model.

Animals↗

Soluble V domain of Nectin-1/HveC enables entry of herpes simplex virus type 1 (HSV-1) into HSV-resistant cells by binding to viral glycoprotein D.

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1(123)), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1(123), approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1(123) did not associate with the cell surface. sNec1(123)-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.

Animals↗

Replication-defective genomic HSV gene therapy vectors: design, production and CNS applications.

Herpes simplex virus (HSV) is a neurotropic double-stranded DNA virus. In an unmodified form it is a human pathogen, causing recurrent cold sores, keratitis and, rarely, severe encephalitis. Elimination of pathogenic functions results in the generation of a valuable gene transfer vector for neurological applications. Replication-defective genomic HSV-based vectors are highly infectious, and efficiently transduce and express transgenes in a broad range of both dividing and non-dividing cells. Clinically relevant yields of clinical grade vector can be produced by growth in cell lines that complement the viral functions that are deleted in the vectors to eliminate pathogenicity. The viral genome is over 150 kb in length and many of the viral genes may be deleted without compromising viral growth in vitro, and therefore large or multiple transgenes can be accommodated within the vectors. The wild-type virus adopts a lifelong latent state in neurons of sensory ganglia. This property can be exploited in the generation of vectors to allow long-term transgene expression in neurons. In this review, we summarize recent progress in the areas of vector development and vector production, and in developing gene transfer therapeutics to treat malignant glioma.

Animals↗

The stable 2.0-kilobase intron of the herpes simplex virus type 1 latency-associated transcript does not function as an antisense repressor of ICP0 in nonneuronal cells.

During latency, herpes simplex virus expresses a unique set of latency-associated transcripts (LATs). As the 2.0-kb LAT intron is complementary to, and overlaps, the 3' end of the ICP0 transcript, it has been suggested that the stable LAT intron might function as an antisense repressor of ICP0 expression. We tested this hypothesis in cell culture by dissociating cis- and trans-acting effects of the 2.0-kb LAT, using a series of complementary strategies. Initially, we constructed 293T cell lines that stably express the nuclear 2.0-kb LAT intron to determine whether LAT accumulation in trans affects ICP0 expression. ICP0 mRNA and protein expression profiles were studied (i) following infections with a viral mutant containing wild-type LAT and ICP0 sequences but having deletions of other immediate-early (IE) genes, thus preventing the progression of viral early gene expression, (ii) at early time points after infection with wild-type virus, before viral LAT expression, and (iii) by plasmid transfections. Northern and Western blot analysis showed that trans expression of the 2.0-kb LAT intron does not affect ICP0 mRNA expression, stability, accumulation, splicing, or translation. In addition, suppression of viral replication by overexpression of the 2.0-kb LAT, which has been detected previously in neuronal cell lines, was not found in these nonneuronal cell lines. However, deletion of the latency-active promoter (LAP) region of the virus resulted in overexpression of IE genes, which occurred soon after infection, before viral LAT expression had commenced. This was not complemented by the expression of LAT in trans, suggesting that the LAP deletion affected transcriptional regulation of the IE genes in cis. We conclude that the function of the highly conserved LAT intron is unlikely to involve a direct-acting anti-ICP0 antisense mechanism but that the LAT region could affect ICP0 mRNA expression from the viral genome.

Cell Line↗

A- and B-utrophin have different expression patterns and are differentially up-regulated in mdx muscle.

Duchenne muscular dystrophy (DMD) is a fatal childhood disease caused by mutations that abolish the expression of dystrophin in muscle. Utrophin is a paralogue of dystrophin and can functionally replace it in skeletal muscle. A method to induce utrophin up-regulation in muscle should therefore be therapeutically useful in DMD. We have previously shown that there are two full-length utrophin mRNA species: A and B. Here we describe the generation and characterization of antibodies specific to A- and B-utrophin. We show that both mRNA isoforms are translated into full-length proteins, which have very different expression patterns. B-utrophin is expressed in vascular endothelial cells; A-utrophin is expressed at the neuromuscular junction, choroid plexus, pia mater, and renal glomerulus. We have analyzed the expression of A- and B-utrophin protein and RNA in dystrophin-deficient tissues. We conclude that (i) the previously described expression patterns of utrophin represent a composite of A- and B-utrophin, (ii) A- but not B-utrophin is up-regulated in dystrophin-deficient striated muscle, and (iii) this up-regulation occurs post-transcriptionally with an additional transcriptional component in skeletal muscle. These results have important implications for understanding the biology of utrophin and are crucial for future studies aiming to effect its therapeutic up-regulation in DMD patients.

Animals↗

Muscular dystrophy--reason for optimism?

Characterization of the mechanisms underlying various types of muscular dystrophy has been an outstanding triumph of molecular biology. Increasing clarification of the aberrant cellular processes responsible for these conditions may ultimately permit the development of effective means for molecular intervention, allowing correction of the abnormal cellular physiology that results in the dystrophic phenotype.

Dystrophin↗

Replication-defective genomic herpes simplex vectors: design and production.

Herpes simplex virus (HSV) may be engineered to produce flexible and efficient gene delivery vectors. Recent advances in vector design and production have built on increasing understanding of the basic biology of HSV to minimise vector toxicity and exploit viral features that give rise to lifelong latent infection in the nervous system. In addition, the emerging picture of viral cell entry has allowed early steps to be taken towards targeting viral entry to predetermined cellular subsets. Recent work has established sound principles for the straightforward production of large-scale pure preparations of vector stocks for clinical applications.

DNA, Recombinant↗

Gene delivery using herpes simplex virus vectors.

Herpes simplex virus (HSV) is a neurotropic DNA virus with many favorable properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells. Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans, enabling straightforward production of high-titre pure preparations of non-pathogenic vector. The genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime. Transduction with replication-defective vectors causes a latent-like infection in both neural and non-neural tissue; the vectors are non-pathogenic, unable to reactivate and persist long-term. The latency active promoter complex can be exploited in vector design to achieve long-term stable transgene expression in the nervous system. HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed preliminary steps to be taken towards targeting the tropism of HSV vectors. Using replication-defective HSV vectors, highly encouraging results have emerged from recent pre-clinical studies on models of neurological disease, including glioma, peripheral neuropathy, chronic pain and neurodegeneration. Consequently, HSV vectors encoding appropriate transgenes to tackle these pathogenic processes are poised to enter clinical trials.

Animals↗