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S Chamberlain

Publications and source records attributed to S Chamberlain.

At least 19 recordsLinked to original sources
PubMed2001

Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis.

We have generated and characterised transgenic mice that contain the entire Friedreich's ataxia gene (FRDA) within a human YAC clone of 370 kb. In an effort to overcome the embryonic lethality of homozygous Frda knockout mice and to study the behaviour of human frataxin in a mouse cellular environment, we bred the FRDA YAC transgene onto the null mouse background. Phenotypically normal offspring that express only YAC-derived human frataxin were identified. The human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and it was correctly processed and localised to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function, together with some increase in citrate synthase and aconitase activities. Thus, we have demonstrated that human frataxin can effectively substitute for endogenous murine frataxin in the null mutant. Our studies are of immediate consequence for the generation of Friedreich's ataxia transgenic mouse models, and further contribute to the accumulating knowledge of human-mouse functional gene replacement systems.

Animals↗
PubMed2001

Temporary ischaemia of the scaphoid in a child.

We describe a case of a 12-year-old boy who developed acute ischaemia of the entire scaphoid following simple trauma to the wrist. No fracture was evident; however, MRI and bone scintigraphy at the time of the injury revealed complete ischaemia of the scaphoid. A repeat MRI at 6 months showed evidence of incomplete revascularisation of the scaphoid.

Child↗
PubMed2001

Invasiveness of fibroblasts from experimental cholesteatomas.

HYPOTHESIS: Cultured fibroblasts derived from experimental gerbil cholesteatoma tissue exhibit an invasive phenotype in comparison with normal fibroblasts. BACKGROUND: Aural cholesteatomas are enlarging accumulations of keratin debris caused by keratinizing squamous epithelium in the middle ear. They characteristically result in the destruction of adjacent tissues, specifically bone erosion. The mechanisms by which cholesteatomas relentlessly invade the structures of the temporal bone are varied, but it has been suggested that one factor contributing to the aggressive nature of cholesteatomas is the transformation of resident fibroblasts into an invasive phenotype. METHODS: The ability of cultured normal and cholesteatoma fibroblasts to invade a basement membrane matrix in a Boyden chamber assay was examined. RESULTS: Less than 1% of gerbil fibroblasts invaded the matrix, compared with almost 10% of the invasive HT-1080 fibrosarcoma cells. Normal and cholesteatoma fibroblasts did not differ from each other in their invasive potential. CONCLUSION: Normal fibroblasts and fibroblasts from induced cholesteatomas do not exhibit the invasive phenotype characteristic of true neoplastic cells.

Animals↗
PubMed2001

Chronic pain.

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Chronic Disease↗
PubMed2000

Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive disorder with a frequency of 1 in 50 000 live births. In 97% of patients it is caused by the abnormal expansion of a GAA repeat in intron 1 of the FRDA gene on chromosome 9, which encodes a 210 amino acid protein called frataxin. Frataxin is widely expressed and has been localized to mitochondria although its function is unknown. We have investigated mitochondrial function, mitochondrial DNA levels, aconitase activity and iron content in tissues from FRDA patients. There were significant reductions in the activities of complex I, complex II/III and aconitase in FRDA heart. Respiratory chain and aconitase activities were decreased although not significantly in skeletal muscle, but were normal in FRDA cerebellum and dorsal root ganglia, although there was a mild decrease in aconitase activity in the latter. Mitochondrial DNA levels were reduced in FRDA heart and skeletal muscle, although in skeletal muscle this was paralleled by a decline in citrate synthase activity. Increased iron deposition was seen in FRDA heart, liver and spleen in a pattern consistent with a mitochondrial location. The iron accumulation, mitochondrial respiratory chain and aconitase dysfunction and mitochondrial DNA depletion in FRDA heart samples largely paralleled those in the yeast YFH1 knockout model, suggesting that frataxin may be involved in mitochondrial iron regulation or iron sulphur centre synthesis. However, the severe deficiency in aconitase activity also suggests that oxidant stress may induce a self-amplifying cycle of oxidative damage and mitochondrial dysfunction, which may contribute to cellular toxicity.

Aconitate Hydratase↗
PubMed1999

Insertion of expanded CAG trinucleotide repeat motifs into a yeast artificial chromosome containing the human Machado-Joseph disease gene.

Machado-Joseph disease or spinocerebellar ataxia 3 (SCA3) is a progressive neurodegenerative disorder caused by pathological expansion of a trinucleotide repeat motif present within exon 4 of the MJD1 gene. Previous attempts to create a transgenic animal model have failed to produce a neurological deficit truly representative of the disease phenotype. This appears to be the result of inappropriate expression of the mutant protein in neuronal populations generally spared in the disease state. Introduction of a human disease gene in the context of a yeast artificial chromosome clone containing endogenous regulatory elements would enhance the potential for correct tissue/cell-specific expression at physiological levels. We report the introduction of expanded CAG repeat motifs into a 250kb yeast artificial chromosome clone spanning the MJD1 locus using two rounds of homologous recombination. Transformants exhibited both expansions and contractions of the motif with alleles ranging in size from 48 to 84 repeat units. The availability of these clones for modelling of the disease in transgenic animals should allow elucidation of the role of repeat length in the phenotypic spectrum of the disease.

Ataxin-3↗
PubMed1999

Development of novel benzimidazole riboside compounds for treatment of cytomegalovirus disease.

Benzimidazole ribosides are a new class of compounds with novel mechanisms of action against CMV. One compound in this series, BDCRB, inhibits CMV DNA processing by the UL89 gene product (putative terminase), but rapid metabolism to an inactive compound makes it unsuitable for development as a medicine. Another benzimidazole analogue, 1263W94, has many characteristics that make it an attractive candidate for development, including high potency in vitro, selectivity, good oral bioavailability, and lower toxicity than therapies currently available for treatment of CMV disease. Initial clinical trials have provided encouraging results, including good tolerability and linear pharmacokinetics over a wide dose range. Ongoing and planned clinical trials that will study the safety and tolerability of repeated dosing and evaluate the in vivo antiviral activity and ocular penetration of 1263W94, will help to determine the potential of this drug as an improved therapy for CMV disease.

Antiviral Agents↗
PubMed1999

Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin.

Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system. The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin. The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, beta-galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo. We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine [poly(K)] to enable binding of DNA. We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma x glioma mouse/rat hybrid cell line) and F98 (a glioma cell line). Transfection was dependent on HC and was neuronal cell type specific. HC may prove a useful targeting ligand for future neuronal gene therapy.

Cation Exchange Resins↗
PubMed1999

A family with pseudodominant Friedreich's ataxia showing marked variation of phenotype between affected siblings.

A family with pseudodominant Friedreich's ataxia is described showing marked variation of phenotype between affected siblings. The mother of this family (III-3) developed a spastic ataxic tetraplegia with neuropathy at 34 years of age; her husband, who was unrelated, was clinically normal. Of their nine children, two (IV-2, IV-3), including one with multiple sclerosis (IV-3), developed a mild spinocerebellar degeneration in the third decade. Three in their late 20s had an asymptomatic spinocerebellar degeneration (IV-4, IV-5, IV-6) and one was confined to a wheelchair at 15 years with typical Friedreich's ataxia (IV-9). Three other siblings (IV-1, IV-7, IV-8) were clinically normal. The father proved to be heterozygous for the triplet repeat expansion at the Friedreich's ataxia locus and all clinically affected members were homozygous for alleles in the expanded size range. This family confirms that homozygote-heterozygote mating is the genetic basis for some families with apparent autosomal dominant Friedreich's ataxia.

Adult↗
PubMed1998

The granulin/epithelin precursor abrogates the requirement for the insulin-like growth factor 1 receptor for growth in vitro.

3T3 cells null for the type 1 insulin-like growth factor receptor are refractory to stimulation by a variety of purified growth factors that are known to be required for the stimulation of other 3T3 cells. However, these cells, known as R- cells, grow in serum-supplemented medium and also in media conditioned by certain cell lines. We report here the purification of a growth factor that stimulates DNA synthesis (and growth) of R- cells. The growth factor, purified to homogeneity by SDS-polyacrylamide gel electrophoresis, was identified as the granulin/epithelin precursor by an accurate determination of the masses of endoproteinase Lys-C peptides using matrix-assisted laser desorption ionization mass spectrometry, followed by a data base search. The granulin/epithelin precursor is a little known growth factor, secreted by a variety of epithelial and hemopoietic cells. It is at present the only purified growth factor that can stimulate the growth of mouse embryo fibroblasts null for the type 1 insulin-like growth factor receptor.

3T3 Cells↗
PubMed1998

A clinicogenetic analysis of six Indian spinocerebellar ataxia (SCA2) pedigrees. The significance of slow saccades in diagnosis.

Clinical revaluation and genetic analysis of six Indian pedigrees, segregating autosomal dominant cerebellar ataxia, slow saccades and peripheral neuropathy, has been undertaken, and expansion at the spinocerebellar ataxia 2 (SCA2) locus was confirmed in 14 affected family members. These families became available from 31 phenotypically similar families seen over the years. In common with other neurodegenerative disorders resulting from expansion of a CAG trinucleotide repeat motif, an inverse correlation between repeat size and age at onset and severity is observed, although the size range (36-45 repeat units) for the expanded alleles is comparatively limited. Saccadic velocity was reduced in all our patients, even in the early stages of the disease. The observation of slow saccades in affected individuals has been proposed previously as an important diagnostic criterion serving to distinguish the SCA2 phenotype. This is now confirmed in a retrospective study of the clinical literature, facilitated by the cloning of the SCA2 gene and the subsequent genetic analysis of families segregating this phenotype. We therefore argue that the clinical appraisal of 'ophthalmoplegia' be subject to more precise definition, as differentiation between the various types of ocular dysfunction can be an important adjunct to diagnosis.

Adolescent↗
PubMed1997

A sequence-ready physical map of a region of 12q24.1.

We developed a sequence-ready map of a part of human chromosome 12q24.1. We utilized a number of sequence-tagged site (STS) markers from 12q24.1 to screen large insert bacterial chromosome libraries and a chromosome 12-specific cosmid library. The clones were assembled into contiguous sets (contigs) by STS-content analysis. Contigs were extended by obtaining end sequences of bacterial clones, generation of additional STSs, rescreening the libraries, and screening the additional clones for the presence of STSs. The resulting contig covers nearly 2 Mb of DNA and provides an average marker resolution of 16 kb. Based on the STS content, we developed fingerprints of a subset of clones. The STS content and fingerprint data allowed us to define a minimal tiling path of clones. These clones are being used to sequence this part of chromosome 12. This contig contains the Ataxin 2 gene, and it covers the interval harboring the gene responsible for Darier disease.

Ataxins↗
PubMed1997

Exon-intron structure of a 2.7-kb transcript of the STM7 gene with phosphatidylinositol-4-phosphate 5-kinase activity.

The STM7 gene encodes a novel phosphatidylinositol-4-phosphate 5-kinase (PtdInsP 5-kinase) that is subject to alternative splicing and developmental control. We have recently presented data indicating that several splice variants of STM7 incorporate elements of the X25 sequence, previously implicated in the pathogenesis of Friedreich's ataxia by the detection of an intronic GAA repeat expansion as the predominant mutation in affected individuals. We now report the exon-intron structure of STM7.I and primer sequences designed to facilitate full characterization, including details relating to a novel exon (STM7; exon 17) derived from the 3'-UTR of the PRKACG gene. The detection of a mutation(s) within these exons would provide additional support for the hypothesis that a defect in phosphoinositide metabolism gives rise to the disease phenotype.

Alternative Splicing↗
PubMed1997

Polycystin expression is temporally and spatially regulated during renal development.

Mutations in PKD1 cause autosomal dominant polycystic kidney disease (ADPKD), a common genetic disease in which cysts form from kidney tubules. The predicted product of this gene is a novel protein with cell-adhesive and membrane-spanning domains. To test the hypothesis that polycystin, the product of the PKD1 gene, is a cell adhesion molecule, we raised antibodies against peptides derived from the unduplicated, membrane-spanning portion of the predicted amino acid sequence. These antibodies recognized membrane-associated polypeptides of 485 and 245 kDa in human fetal kidney homogenates. Expression was greater in fetal than adult kidney by both Western blot analysis and immunofluorescence. In fetal kidney, polycystin was localized to the plasma membranes of ureteric bud and comma and S-shaped bodies. However, in more mature tubules in fetal kidney, in adult kidney, and in polycystic kidney, the majority of polycystin staining was intracellular. The temporal and spatial regulation of polycystin expression during renal development lead us to speculate that polycystin may play a role in nephrogenesis.

Adult↗
PubMed1997

Human androgen receptor expression in prostate cancer following androgen ablation.

OBJECTIVE: Metastatic prostate cancer kills patients because their tumor cells fail to respond to combined androgen blockade (CAB) or respond and then relapse. To understand the molecular basis of androgen-insensitive growth of prostate tumor cells, we evaluated changes in human androgen receptor gene (hAR) mRNA levels in patients with prostate cancer treated with CAB. METHODS: The study was carried out using quantitative reverse-transcriptase polymerase chain reaction analysis. The level of hAR mRNA were compared to serum prostate-specific antigen and the mutant status of p53 in the tumor. RESULTS: hAR was expressed in 44 of 46 tumors from untreated patients, as opposed to 30 of 45 from those who had received CAB (p = 0.001). These 30 were from 8 of 9 stage D patients and from 22 of 36 patients on downsizing CAB therapy prior to radical prostatectomy. Expression was most often seen in high stages (56% of stage B vs. 89% of stage D) and high grades (52% of Gleason 3-7 vs. 92% of Gleason 8-10, p = 0.015). No tumor with a missense p53 mutation had hAR expression following CAB. Twenty-two patients following CAB were found to have undetectable serum prostate-specific antigen levels, while their tumor expressed hAR. CONCLUSIONS: hAR expression after CAB is seen preferentially in high-grade, high-stage tumors, the type of prostate carcinomas that fail to have a durable remission. Undetectable serum prostate-specific antigen from tumors that remain hAR positive may predict relapse after hormonal ablative therapy.

Androgen Antagonists↗