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Biomedical subjects

D Wells

Publications and source records attributed to D Wells.

At least 55 records · Page 3Linked to original sources
PubMed1998

Expression and functional analysis of mouse EXT1, a homolog of the human multiple exostoses type 1 gene.

Hereditary multiple exostoses (EXT) is a genetically heterogeneous, autosomal dominant skeletal disorder. The gene for EXT1 maps to human chromosome 8q24.1 and encodes an evolutionary conserved protein that is a member of a multigene family. The mouse homolog of human EXT1 protein is 99% similar to its human counterpart. Here, we present the expression profiles of the mouse EXT1 gene. EXT1 mRNA is initially expressed at 6.5 days post-coitum (d.p.c.), which coincides with gastrulation of the mouse embryo. Whole mount in situ hybridization with 10.5 to 12.5 d.p.c. mouse embryos showed a high level of expression of EXT1 mRNA in developing limb buds. Epitope tagging experiments revealed the endoplasmic reticulum localization of EXT1 protein. This localization was consistent with a hydrophobic stretch of amino acids present at the N-terminal end of the EXT1 protein. These results provide novel information on the function of EXT1 and the etiology of hereditary multiple exostoses.

3T3 Cells↗
PubMed1998

Helminth levels of working donkeys kept under different management systems in the Moretele 1 district of the North-West Province, South Africa.

In Southern Africa, where 150,000 working donkeys provide an important alternative to mechanisation in resource-poor communities, very little is known about their helminth status, or about the impact of helminths on their work output. The aim of this study was to investigate the helminth status of working donkeys under different management systems. Donkey owners in three different areas (one rural and two semi-rural) of the Moretele 1 district of North-West Province, South Africa, were visited and structured interviews were used to assess the management systems under which the donkeys were kept. Faecal samples were collected from 93 donkeys in the study once a month for 14 months. Faecal samples were analysed for nematode and trematode eggs and cultured to produce third-stage larvae for the identification of the nematode species. Final comparisons between management system subgroups, as well as between areas, age groups and sexes were made. Four management systems were identified. (1) The first system identified consisted of donkeys which were kept in a small yard at all times. They were fed hay but no supplementary food. (2) The second system consisted of donkeys which were allowed to roam freely around the village most of the time and rounded up and held in an enclosure when needed for work. (3) The third system was identical to the second management system except that the donkeys were given supplementary food during winter. (4) The fourth system was only found in the one area where each owner owned 10 ha of land and here the donkeys were allowed to roam freely on the owner's land and brought into enclosures prior to working. Helminth species composition and faecal egg count numbers differed between these four systems. The main difference noted was that donkeys from management system one showed significantly higher numbers of strongyle eggs and higher percentages of some of the strongyle larvae. Management system two had a higher Strongyloides mean egg count and prevalence than the other areas. Parascaris and Gastrodiscus egg counts differed between all four systems. Since the results showed differences in the number and species of helminths in donkeys kept under the four management systems, suggestions are made as to which management system would facilitate reduction of helminth parasites in the animals. Although supplementary feeding in Moretele 1 is fairly rare, it would seem that donkeys which do have access to better food resources have lower egg counts than donkeys on limited grazing.

Age Distribution↗
PubMed1998

High throughput liquid chromatography/mass spectrometry bioanalysis using 96-well disk solid phase extraction plate for the sample preparation.

The 96 well solid phase extraction (SPE) operated by robot in the LC/MS/MS bioanalysis offered rapid sample preparation for drugs and metabolites in biological matrices, based on simultaneous extraction of 96 samples. The use of a disk as sorbent in the 96 well plate further improved the performance of SPE and allowed for small elution volumes, making it possible to 'dilute and shoot" after SPE elution. In this study, a 96 well plate (Empore) was developed, characterized and optimized for several pharmaceutical compounds. In addition, a robot (MultiProbe) was modified to automate the 96 well plate operation. Examples were given to illustrate the major differences of using 96 well disk plate SPE in the method development as compared to the traditional SPE. This technology has been successfully used to support many clinical studies. Typically, a batch of 96 samples were prepared in 1-1.5 hours unattended (except for the replacement of a collection plate). Considerable savings in disposable supplies were also noted.

Anti-Asthmatic Agents↗
PubMed1998

CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses.

Long-term changes in synaptic efficacy may require the regulated translation of dendritic mRNAs. While the basis of such regulation is unknown, it seemed possible that some features of translational control in development could be recapitulated in neurons. Polyadenylation-induced translation of oocyte mRNAs requires the cis-acting CPE sequence and the CPE-binding protein CPEB. CPEB is also present in the dendritic layers of the hippocampus, at synapses in cultured neurons, and in postsynaptic densities of adult brain. alpha-CaMKII mRNA, which is localized in dendrites and is necessary for synaptic plasticity and LTP, contains two CPEs. These CPEs are bound by CPEB and mediate polyadenylation-induced translation in injected Xenopus oocytes. In the intact brain, visual experience induces alpha-CaMKII mRNA polyadenylation and translation, suggesting that this process likely occurs at synapses.

Animals↗
PubMed1998

Postgraduate education in clinical forensic medicine: a graduate diploma.

The absence of a formal postgraduate course has had a significant impact on the development of clinical forensic medicine. Increasing scrutiny by the criminal justice system and recognition of the specialized nature of the work have led to the need for an academic basis for the discipline. Monash University in Victoria, has developed a course leading to a Graduate Diploma in Forensic Medicine. Combining principles and practice taught largely by distance education methods, the programme will enhance practitioners' knowledge and skills in clinical forensic medicine.

Journal Article↗
PubMed1998

Preimplantation genetic diagnosis of inherited cancer: familial adenomatous polyposis coli.

PURPOSE: Our purpose was to achieve preimplantation genetic diagnosis (PGD) of the dominant cancer predisposition syndrome, familial adenomatous polyposis coli (FAPC), as an alternative to prenatal diagnosis. METHODS: The affected patient was superovulated and oocytes were retrieved and fertilized by intracytoplasmic sperm injection (ICSI). Two cells were biopsied from each embryo and the whole genome was amplified by primer extension preamplification (PEP). Nested PCR was then used to amplify two APC fragments: one including the APC mutation site and the other an informative intragenic polymorphism. Both were detected by simultaneous single-strand conformation polymorphism and heteroduplex analysis. RESULTS: Four normally fertilized embryos were biopsied on day 3 post ICSI, and two cells were successfully removed from each embryo. Following PEP the APC mutation was successfully amplified in 7 of 8 cells, and the polymorphism in 6 of 8 cells. The APC mutation was detected in three embryos. This result was confirmed by identification of the mutation associated polymorphism in two cases. A single embryo was diagnosed as homozygous normal for the mutation and the polymorphism in both cells sampled. This unaffected embryo was transferred to the mother, but no pregnancy resulted. CONCLUSIONS: We report here the first diagnosis of a cancer predisposition syndrome in human preimplantation embryos. Our results indicate that difficulties associated with single-cell PCR, allele-specific amplification failure in particular, need not prevent preimplantation diagnosis of diseases with a dominant mode of inheritance, provided appropriate strategies are applied.

Adenomatous Polyposis Coli↗
PubMed1998

Mutations of adenomatous polyposis coli (APC) gene are uncommon in sporadic desmoid tumours.

Desmoids are locally aggressive, non-metastasizing soft-tissue tumours, whose aetiology is still unclear. In patients affected with familial adenomatous polyposis (FAP), the incidence of desmoids is much higher than in the general population. The APC gene, which is responsible for FAP, is involved in the development of desmoids associated with this syndrome. In this study 16 sporadic and four FAP-related desmoids were analysed in order to investigate the possible involvement of APC in non-syndromic cases also. The 5' end (exons 1-11) and the coding portion of exon 15 of APC were screened using the in vitro synthesized-protein assay (IVSP). Exons 5, 6, 8-14, and a region of exon 15 spanning codons 1036-1634 were investigated by single-strand conformation polymorphism (SSCP) analysis. APC germline mutations were identified in all FAP patients, but not in sporadic cases. Somatic mutations were found in three FAP-associated desmoids (75%) and two sporadic tumours (12.5%). In one of the latter cases, both alleles were affected. These findings indicate a limited role of the gene in the development of desmoid tumours outside FAP.

Adenomatous Polyposis Coli↗
PubMed1998

Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification.

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for ensuring the genetic health of offspring born to families affected by inherited disease. This paper sets out to review current protocols for the diagnosis of single gene defects in human preimplantation embryos. These methods, which depend on DNA amplification using PCR, are subject to a variety of pitfalls, such as allele dropout (ADO), contamination and reduced amplification efficiency. Advances in single cell DNA amplification, such as improved multiplex PCR protocols, fluorescent-PCR and whole genome amplification (WGA), can be applied to address some of these problems. Different PGD strategies are discussed in the context of their clinical application.

DNA Mutational Analysis↗
PubMed1997

Structure and chromosomal mapping of the mouse P2X3 gene.

P2X3 is one of seven cloned ATP-gated non-selective cation channels. We have isolated a full-length mouse P2X3 gene from a phage lambda-129/Sv genomic library. The gene consists of 12 exons spanning a locus of approximately 40 kb. No significant similarities have been found between the genomic organisation of the mouse P2X3 gene and genes encoding other ion channels. The encoded mouse P2X3 protein consists of 397 amino acids and shows 99% identity with rat P2X3. Using RNase protection and primer extension assays, multiple transcription initiation sites have been mapped in the mouse P2X3 promoter to a region 162-168 bp upstream of the translation initiation codon. The P2X3 gene has been mapped to mouse chromosome 2p by fluorescence in situ hybridisation. The RAG locus-associated gene T160 is located 1.8 kb upstream of the transcription start site of mouse P2X3 gene. The promoter region of the mouse P2X3 gene lacks a conventional TATA and CCAAT consensus sites, and initiator elements. P2X3 is the first member of the P2X gene family to be completely characterised.

Amino Acid Sequence↗
PubMed1997

Automated sample preparation using membrane microtiter extraction for bioanalytical mass spectrometry.

The development and application of membrane solid phase extraction (SPE) in 96-well microtiter plate format is described for the automated analysis of drugs in biological fluids. The small bed volume of the membrane allows elution of the analyte in a very small solvent volume, permitting direct HPLC injection and negating the need for the time consuming solvent evaporation step. A programmable liquid handling station (Quadra 96) was modified to automate all SPE steps. To avoid drying of the SPE bed and to enhance the analytical precision a novel protocol for performing the condition, load and wash steps in rapid succession was utilized. A block of 96 samples can now be extracted in 10 min., about 30 times faster than manual solvent extraction or single cartridge SPE methods. This processing speed complements the high-throughput speed of contemporary high performance liquid chromatography mass spectrometry (HPLC/MS) analysis. The quantitative analysis of a test analyte (Ziprasidone) in plasma demonstrates the utility and throughput of membrane SPE in combination with HPLC/MS. The results obtained with the current automated procedure compare favorably with those obtained using solvent and traditional solid phase extraction methods. The method has been used for the analysis of numerous drug prototypes in biological fluids to support drug discovery efforts.

Autoanalysis↗
PubMed1997

Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein-DNA interactions.

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to region 5' and 3' of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3' end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

Animals↗
PubMed1997

Single cell detection of beta-thalassaemia mutations using silver stained SSCP analysis: an application for preimplantation diagnosis.

Although prenatal diagnosis has reduced the number of beta-thalassaemia births, pregnancy termination is still unacceptable for many couples. Preimplantation genetic diagnosis carried out on the third day after in-vitro fertilization offers an alternative. Here we describe the detection of selected beta-thalassaemia mutations in intron I at the single cell level by the application of nested polymerase chain reaction (PCR) and silver stained single strand conformation polymorphism (SSCP) analysis. A total of 294 single somatic cells of different types was amplified with 96% success and all tested mutations in homozygous and heterozygous form were identified correctly. None of the heterozygous or compound heterozygous samples showed any allele-specific amplification failure after the beta-globin gene amplification from single cells. To assess the efficiency of nested PCR on single blastomeres prior to clinical application, 10 single blastomeres were amplified and gave the expected normal pattern when analysed by SSCP. The main advantage of SSCP, particularly for preimplantation diagnosis, is that it allows the direct visualization of each allele and provides a simple means of assessing allele-specific amplification failure. Our results show that the combination of nested PCR and automated silver stained SSCP analysis offers exceptional resolution, accuracy and speed which are essential for preimplantation diagnosis.

Alleles↗
PubMed1997

Regional assignment of 64 human gene transcripts on chromosome 8.

A panel of somatic cell hybrids specific for human chromosome 8 was used to localize 64 expressed sequence-tagged site (ESTS) markers to six individual regions by PCR amplification. Nine ESTS correspond to 8 known human genes and 6 others show similarities with vertebrate genes, whereas the remaining 49 ESTS markers correspond to novel genes with no database similarities. These gene transcript markers will contribute to the developing physical and expression maps of chromosome 8, and to the search for candidate genes for various human diseases.

Animals↗
PubMed1997

Isolation of the mouse cDNA homologous to the human EXT1 gene responsible for Hereditary Multiple Exostoses.

We have cloned and sequenced the mouse cDNA homologous to the human Hereditary Multiple Exostoses (EXT1) gene. The mouse homolog shows 94% similarity at the nucleotide level and 99% similarity at the protein level compared to the human gene. The 5' UTRs are unusually conserved for non-coding sequences showing 94% similarity compared to 80% for the 3' UTRs. The high level of evolutionary conservation between the EXT1 proteins as well as the 5' UTR suggests that each plays an important and related role in both species.

Amino Acid Sequence↗