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Biomedical subjects

J G Dickson

Publications and source records attributed to J G Dickson.

At least 19 recordsLinked to original sources
PubMed2006

Gene therapy progress and prospects: Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1/3500 male births. There is currently no effective treatment, but gene therapy approaches are offering viable avenues for treatment development. The last 10 years have seen the development of a number of strategies and tools for muscle gene therapy. However, the major hurdle has been the inability to deliver vectors at high enough efficiency via a systemic route. The last 2-3 years (reviewed here) have seen unrivalled progress in efficient systemic delivery of viral and non-viral gene transfer agents and antisense oligonucleotides. This progress, coupled with the successful completion of the first gene therapy clinical trial for DMD, has led to three more clinical trials planned for the immediate future.

Animals↗
PubMed2001

Gene correction of the apolipoprotein (Apo) E2 phenotype to wild-type ApoE3 by in situ chimeraplasty.

Apolipoprotein (apo) E is a polymorphic plasma protein, synthesized mainly by liver. Here, we evaluate whether synthetic DNA-RNA oligonucleotides (chimeraplasts) can convert a dysfunctional isoform, apoE2 (C --> T, R158C), which causes Type III hyperlipidemia and premature atherosclerosis, into apoE3. First, we treated recombinant Chinese hamster ovary cells stably secreting apoE2 with a 68-mer apoE2 to apoE3 chimeraplast. About one-third of apoE2 was converted to apoE3, and the repair was stable through 12 passages. Subcloning treated cells produced both apoE2 and apoE3 clones. Direct sequencing and reverse transcription polymerase chain reaction confirmed the genotype, whereas phenotypic change was verified by isoelectric focusing and immunoblotting of secreted proteins. Second, we established that the APOE2 gene can be targeted both in vivo, using transgenic mice overexpressing human apoE2, and in chromosomal context, using cultured lymphocytes from a patient homozygous for the epsilon2 allele. We conclude that chimeraplasty has the potential to convert the apoE2 mutation in patients with Type III hyperlipidemia to apoE3.

Amino Acid Substitution↗
PubMed2000

Technology evaluation: AAV factor IX gene therapy, Avigen Inc.

Avigen has developed a recombinant adeno-associated viral vector expressing human blood-coagulation Factor IX (F.IX) for the potential treatment of hemophilia B. In a phase I clinical trial being conducted at The Children's Hospital of Philadelphia and Stanford University Medical Center, the vector, AAV-CMV-hF.IX (Coagulin-B), was injected at a low dose into three patients with severe hemophilia B. No evidence of toxicity, germline transmission of vector sequences, or formation of inhibitory antibodies against F.IX was observed, and in two of the three patients there was an indication of a modest clinical response.

Animals↗
PubMed1997

Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy.

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.

Animals↗
PubMed1991

Choline acetyltransferase messenger RNA expression in developing and adult rat brain: regulation by nerve growth factor.

The polymerase chain reaction (PCR) was used to develop a method for detection and relative quantification of the choline acetyltransferase (ChAT) mRNA in neonatal and adult rat CNS. Oligonucleotide primers derived from a porcine ChAT cDNA sequence were used in coupled reverse transcriptase (RT)-PCR to amplify a cDNA sequence of 206 bp which arises in a cycle- and RNA-dependent manner and which hybridizes with both an internal oligonucleotide and a ChAT cDNA probe. ChAT mRNA was detected in spinal cord, septal area, striatum, cortex and hippocampus but not in cerebellum and cardiac or skeletal muscle. In the septal area, relative quantitative evaluation of ChAT mRNA levels by RT-PCR indicates that this transcript is developmentally regulated and increased following intracerebral administration of nerve growth factor (NGF) to both neonatal and young adult rats. This suggests that the increases of ChAT activity observed in basal forebrain during development or after NGF administration are, at least in part, associated with an increase in corresponding levels of mRNA.

Aging↗
PubMed1988

Expression of membrane antigens in myotonic dystrophy.

Muscle biopsies from a series of myotonic dystrophy patients were analysed for expression of the nerve regulated gene products neural cell adhesion molecule (N-CAM) and 5.1H11. All eight biopsy specimens tested strongly expressed N-CAM and 5.1H11 as assessed by indirect immunofluorescence analysis. These results can be compared with those of Renaud et al (Nature, 1986;319:678) that show apamin binding to be a good marker of myotonic dystrophy muscle membranes. We suggest that in myotonic dystrophy a number of nerve regulated membrane markers are precociously expressed by innervated myofibres and that these are likely to be secondary manifestations resulting from an unidentified primary defect.

Adult↗
PubMed1987

Nerve growth factor-induced changes in neural cell adhesion molecule (N-CAM) in PC12 cells.

The effects of nerve growth factor (NGF) on the expression of neural cell adhesion molecule (N-CAM) in PC12 cells were determined. A quantitative immunoassay was used to show that NGF induces a 4- to 5-fold increase in relative N-CAM levels over a 3-day period. This increase could not be mimicked by cholera toxin suggesting that it is not a simple consequence of morphological differentiation. The changes in N-CAM levels induced by NGF were accompanied by changes in N-CAM molecular forms. The 140-kd N-CAM species is the major N-CAM expressed by naive PC12 cells, while NGF-treated cultures express N-CAM species of 180 kd and 140 kd. Northern analysis showed that naive cells express a 6.7-kd N-CAM mRNA species only, while NGF-treated cultures express both a 6.7-kb and a 7.2-kb transcript. As the 6.7-kb and 7.2-kb mRNAs are alternative spliced transcripts of a single gene, this result shows that NGF can activate a neuron-specific splicing mechanism. This is the first description of control of N-CAM expression by a growth factor.

Adrenal Gland Neoplasms↗
PubMed1987

Skeletal muscle neural cell adhesion molecule (N-CAM): changes in protein and mRNA species during myogenesis of muscle cell lines.

Qualitative and quantitative changes in neural cell adhesion molecule (N-CAM) protein and mRNA forms were measured during myogenesis in G8-1 and C2 cell lines. Indirect immunofluorescence assay showed that N-CAM was constitutively expressed by myoblasts in culture and that myotubes appeared to be stained more strongly. These changes were quantified using a dot blot assay. N-CAM levels increased almost 4-fold in G8-1 cells and 15-fold in C2 cells during myogenesis. The kinetics of accumulation of N-CAM were not coordinate with other muscle markers such as creatine kinase or acetylcholine receptor levels, since N-CAM accumulated significantly ahead of these markers. Immunoblotting showed that myogenesis was not associated with changes in the extent of sialylation of N-CAM. However, distinct changes in desialo forms were observed after neuraminidase treatment. Myogenesis was accompanied by increases in 125- and 155-kD desialo forms with minor changes in 120- and 145-kD forms. Biosynthetic labeling studies showed that myoblasts specifically expressed a transmembrane isoform of 145 kD that was phosphorylated and was down-regulated in myotubes. Pulse-chase analysis of myotubes showed that the 120-kD isoform and an isoform of 145 kD that co-migrated with, but was distinct from, the 145 kD transmembrane isoform of myoblasts were precursors of the 125- and 155-kD isoforms, respectively, that accumulated in myotubes. The 125- and 155-kD isoforms in myotubes are linked to the cell membrane via phosphatidylinositol linkage and can be released by phospholipase C. Indirect immunofluorescence analysis showed that phosphatidylinositol specific phospholipase C specifically released N-CAM from the myotube membrane generating N-CAM-free myotubes, while myoblasts were unaffected by this treatment. Three N-CAM mRNA species were observed in mouse muscle cell lines. Myoblasts were characterized by their expression of 6.7- and 5.2-kb transcripts while myotubes express 5.2- and 2.9-kb transcripts. Thus, myogenesis is qualitatively associated with a down regulation of the 6.7-kb transcript and an up regulation of the 5.2- and 2.9-kb transcript.

Antigens, Surface↗
PubMed1986

Analysis of specific protein synthesis by cultures of motor neuron-enriched cells from embryonic chicken using dual-label two-dimensional gel electrophoresis.

Protein synthesis in motor neuron-enriched and motor neuron-depleted cell cultures from chick embryo spinal cord have been examined by two-dimensional gel electrophoresis in conjunction with dual-label autoradiography. The majority of the 800 or so more abundant cellular proteins examined were found to be commonly expressed by both culture systems. A number of minor protein species were, however, clearly identified as being exclusively synthesized by motor neuron-enriched cell cultures and may thus comprise a small set of motor neuron-specific gene products.

Animals↗
PubMed1986

Human skeletal muscle cells synthesise a neuronotrophic factor reactive with spinal neurons.

Retrograde trophic influences originating in the skeletal musculature have been postulated to be involved in regulating survival and differentiation of embryonic motor neurons and reactive terminal sprouting of mature motor fibres. We have previously described the use of a quantitative immunoassay for neurofilament protein to bioassay in vitro the cell-type-specific neuronotrophic activity of nerve growth factor (NGF) on sensory ganglion neurons. In the present study, the effect of media conditioned by adult human muscle cells (MCM) on the in vitro development of chicken spinal neurons has been studied using a similar approach. Significant increases in neurofilament protein levels in 7-day chicken embryonic spinal cord cultures were found with doses of MCM protein as low as 0.4 microgram/ml, with a dose-response relationship yielding maximal and half-maximal effects at 4 and 1 microgram/ml, respectively. Maximal increases in neurofilament protein levels were associated with an approximate two-fold increase in neuronal cell survival. MCM also induced increases in choline acetyltransferase activity in chick spinal cord cultures. In both the absence and presence of NGF, MCM did not increase neurofilament protein expression in primary cultures of sensory neurons.

Animals↗
PubMed1986

Identification and characterization of neuron-specific and developmentally regulated gene transcripts in the chick embryo spinal cord.

Clones corresponding to neuron-specific and developmentally regulated messenger RNA species in the chick have been isolated from a complementary DNA library prepared using polyadenylated RNA from 7-day embryonic spinal cord. The library was initially screened by differential complementary DNA hybridization procedures for clones identifying polyadenylated RNAs present in embryonic spinal cord but absent from or at low abundance in liver tissue. A high proportion of selected recombinant plasmids were found to identify different RNA species which, although present in 14-day embryonic spinal cord, could not be detected in a corresponding region of the developing chick CNS that is devoid of neuronal cell bodies, the optic nerve. The neuron-specific assignment of these mRNAs within the developing neuroectoderm was confirmed using bulk-isolated neuronal and glial-enriched cell fractions from 7-day embryonic spinal cord. In addition, several distinctive patterns of developmentally regulated expression of neuron-specific messenger RNA species have been observed in the chick spinal cord. The studies lay a foundation for detailed examination of the regional and temporal distribution and control of neuronal gene expression in the chick spinal cord during embryogenesis.

Animals↗
PubMed1986

Effects of amyotrophic lateral sclerosis serum on cultured chick spinal neurons.

We used a quantitative immunoassay to examine the effects of human serum and immunoglobulins on neurofilament protein expression in cultures of chick spinal neurons. Compared with cultures grown in the presence of serum from healthy controls or patients with other neurologic disorders, ALS serum lowered the level of neurofilament proteins. Effects were similar with or without muscle-derived neurotrophic factors; there was no specificity for motor neurons. No neurotoxic activity was found in immunoglobulin fractions, and there was no evidence of circulating antibodies that might neutralize muscle-derived neurotrophic factors or induce cytolysis of spinal neurons.

Adult↗
PubMed1985

Molecular specificity of ganglioside effects on neurite regeneration of sensory neurons in vitro.

Highly purified preparations of individual gangliosides have been tested for their ability to modulate the survival and morphological differentiation of embryonic chick dorsal root ganglion neurons. When added at 48 h to established cultures of nerve growth factor (NGF)-dependent neurons, all ganglioside species tested increased the expression of neurofilament protein. Poly- and trisialogangliosides were more effective than di- or monosialogangliosides. In contrast, neither NGF nor an antiserum against NGF influenced neurofilament protein expression over this period of culture. In addition, ganglioside-induced expression of neurofilament protein was not inhibited by the anti-NGF serum.

Animals↗
PubMed1985

Cellular distribution of 04 antigen and galactocerebroside in primary cultures of human foetal spinal cord.

The distribution of cell-surface 04 antigen and galactocerebroside (GC) was examined by dual-label indirect immunofluorescence assays on live primary cultures of human spinal cord cells dissociated from 8-12 week-old foetal tissue. Oligodendrocytes expressing GC on their surface were present in the cultures at early time points, and all GC-positive cells were found to also express cell-surface 04 antigen. The 04 antigen was found additionally on a further population of GC-negative cells in the spinal cord cultures, which did not react with antibodies to glial fibrillary acidic protein (GFAP), and were distinct from neuronal cells and cell processes which stained with anti-neurofilament antibody. Previous studies in mouse neural cell cultures have shown that 04 antigen-positive cells are direct precursors to GC-bearing oligodendrocytes (Schachner et al. 1982). In the human spinal cord cultures, a rapid decline in the number of cells expressing GC and/or the 04 antigen to a value below 1% was observed during the first 3 days in vitro. The present studies indicate that synthesis of GC occurs in the human spinal cord many weeks before myelination commences in vivo and that GC-negative oligodendrocyte precursors are present simultaneously with more mature GC-positive cells. In addition, it would appear that complex humoral or cellular ingredients may be required for the long-term in vitro maintenance of viable human foetal oligodendrocytes.

Antigens, Surface↗
PubMed1985

Cell survival characteristics and choline acetyltransferase activity in motor neurone-enriched cultures from chick embryo spinal cord.

The effect of muscle extract on cell survival and choline acetyltransferase (ChAT) activity in cultures of enriched cholinergic neurones from 7-day chick embryo spinal cord was examined. When neurones were grown on hydrated collagen gels, considerable cell survival and ChAT activity were obtained even in the absence of tissue extract. These parameters were stimulated twofold in the presence of skeletal muscle extract but not liver or skin extracts. The cholinergic neurotrophic activity was found to be heat- and trypsin-sensitive, nondialysable, and to act in the virtual absence of glial cells. These data are consistent with a retrogradely acting motor neurone trophic activity.

Animals↗
PubMed1985

Ganglioside GM1 does not initiate, but enhances neurite regeneration of nerve growth factor-dependent sensory neurones.

An enzyme-linked immunoadsorbent assay (ELISA) for neurofilament protein was utilised to quantify the effect of exogenous ganglioside on neurite regeneration in cultures of dorsal root ganglion neurones. In contrast to nerve growth factor (NGF), ganglioside GM1 (100 micrograms/ml) failed to support neuronal survival and neurite regeneration as quantified by the ELISA assay and confirmed by morphological criteria. However, the simultaneous presence of GM1 (100 micrograms/ml) and NGF (0.5-5 ng/ml) throughout a 5-day period of culture resulted in an enhancement of previously reported NGF-induced increases in the expression of neurofilament protein. Further, the addition of GM1 (0-200 micrograms/ml) at 48 h in vitro to cultures initially established in the presence of 5 ng/ml NGF substantially increased the subsequent expression of neurofilament protein, this response being both independent of and not potentiated by NGF. The results in the present system suggest that GM1 cannot initiate a programme of neurite regeneration; however, GM1 can enhance this process with the response being secondary to the effect of NGF.

Animals↗
PubMed1984

The effect of nerve growth factor and its antibodies on neurofilament protein expression in primary cultures of sensory and spinal neurons.

An enzyme-linked immunoadsorbent assay has been used to quantify the binding of an antineurofilament monoclonal antibody, RT97, to primary cultures of embryonic chick dorsal root ganglion and spinal cord tissue. In the case of dorsal root ganglion cells a dose-dependent relationship between nerve growth factor (NGF) concentration and the level of RT97 binding was observed. This response could be antagonised by an anti-serum directed against NGF. In contrast, neither NGF nor the antiserum against NGF influenced the binding of RT97 in primary cultures initiated from chick spinal cord.

Animals↗