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S L Smith

Publications and source records attributed to S L Smith.

At least 19 recordsLinked to original sources
PubMed2006

Epigenetic characteristics of cloned and in vitro-fertilized swamp buffalo (Bubalus bubalis) embryos.

Swamp buffalos are becoming endangered due to reproductive inefficiencies. This is of concern because many countries depend heavily on their products. Somatic cell nuclear transfer (SCNT) is a potential strategy for preserving endangered species. To date, SCNT in swamp buffalo has succeeded in the creation of blastocyst embryos. However, development to term of SCNT swamp buffalos is extremely limited, and only 1 live birth has been reported. An abnormal epigenetic mechanism is suspected to be the cause of developmental failure, as is also seen in other species. The DNA methylation and histone acetylation are key players in epigenetic modification and display marked variability during embryonic preimplantation development. Knowledge of epigenetic modifications will aid in solving the developmental problems of SCNT embryos and improving reproductive technology in the swamp buffalo. The objective of this study was to determine the relationship between preimplantation embryonic development and 2 epigenetic patterns, global DNA methylation and histone acetylation, in SCNT and in vitro-fertilized (IVF) swamp buffalo embryos. In addition, we examined the correlations between those 2 mechanisms in the SCNT and IVF swamp buffalo embryos throughout the developmental stages using double immunostaining and quantification of the emission intensities using confocal microscopy. We discovered an aberrant methylation pattern in early preimplantation-stage swamp buffalo SCNT embryos. In addition, greater variability in the DNA methylation levels among nuclei within SCNT embryos was discovered. Hyperacetylation was also observed in SCNT embryos compared with IVF embryos at the 4- and 8-cell stages (P < 0.05). Dynamic changes and interplay between these 2 epigenetic mechanisms could be crucial for embryonic development during the early preimplantation period. The aberrancies uncovered here may contribute to the low efficiency of SCNT.

Animals↗
PubMed2005

Anomalous mRNA levels of chromatin remodeling genes in swamp buffalo (Bubalus bubalis) cloned embryos.

The swamp buffalo (Bubalus bubalis) is a multi-purpose animal in agriculture that is challenged by extinction due to low reproductive efficiency. Nuclear transfer (NT) has been used to preserve special breeds of buffalo, as well as to increase the number of animals. However, cloned buffalo embryos have impaired development, as in other species. To understand the chromatin remodeling activities in cloned embryos and to improve NT technology, we examined the expression profiles of five genes involved in DNA and histone modifications, DNMT1, DNMT3A, DNMT3B, HAT1 and HDAC1, in single swamp buffalo metaphase II oocytes, NT and in vitro fertilized (IVF) embryos from the two-cell to the blastocyst stage, by quantitative real time RT-PCR. We observed similar expression dynamics for all genes studied in the NT and IVF embryos: relatively constant levels of expression for all genes were found from the MII oocyte up to the eight-cell stage; the levels of mRNA for HAT1 and DNMT3B continued to be stably expressed up to the blastocyst stage; while dramatic increases were seen for DNMT3A and HDAC1. Alternatively, the levels of DNMT1 started to decrease at the eight-cell stage. Despite the similarity in the dynamics of gene expression, dramatic differences in the relative levels of these genes between NT and IVF embryos were observed. The expression levels of all DNA modifying genes were higher in the NT embryos than in the IVF embryos at the eight-cell and blastocyst stages. The genes HDAC1 and HAT1 were also expressed significantly higher at the blastocyst stage in the NT embryos. Our results suggested differences in chromatin remodeling between NT and IVF embryos and that lower levels of DNA passive demethylation and higher levels of DNA de novo methylation occurred in the NT embryos. These observations are novel in the species of buffalo, and may be associated with developmental failure of cloned buffalo embryos due to the transcriptional repression effect of most genes studied here.

Acetyltransferases↗
PubMed2005

Overexpression of aurora B kinase (AURKB) in primary non-small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability.

Aurora kinases are key regulators of chromosome segregation during mitosis. We have previously shown by microarray analysis of primary lung carcinomas and matched normal tissue that AURKB (22 out of 37) and AURKA (15 out of 37) transcripts are frequently over-represented in these tumours. We now confirm these observations in a second series of 44 carcinomas and also show that aurora B kinase protein levels are raised in the tumours compared to normal tissue. Elevated levels of expression in tumours are not a consequence of high-level amplification of the AURKB gene. Using a coding sequence polymorphism we show that in most cases (seven out of nine) tumour expression is predominantly driven from one AURKB allele. Given the function of aurora B kinase, we examined whether there was an association between expression levels and genetic instability. We defined two groups of high and low AURKB expression. Using a panel of 10 microsatellite markers, we found that the group showing the higher level of expression had a higher frequency of allelic imbalance (P=0.0012). Analysis of a number of other genes that are strongly and specifically expressed in tumour over normal lung, including SERPINB5, TERT and PRAME, showed marked allelic expression imbalances in the tumour tissue in the context of balanced or only marginally imbalanced relative allelic copy numbers. Our data support a model of early carcinogenesis wherein defects in the process of inactivation of lung stem-cell associated genes during differentiation, contributes to the development of carcinogenesis.

Allelic Imbalance↗
PubMed2005

Impaired parasympathetic response to feeding in ventilated preterm babies.

BACKGROUND: Premature very low birthweight (VLBW) infants are born with an underdeveloped parasympathetic nervous system (PNS) which may limit their ability to respond adequately to feeding and may limit their capacities for extrauterine growth and development. OBJECTIVES: To describe the patterns of autonomic response to feeding and identify relationships between change in heart period variability measures over time with selected infant characteristics. METHODS: Individual growth curve analysis techniques were used to describe the patterns of change over time in sympathetic and parasympathetic tone as measured by low and high frequency heart period power. RESULTS: Sixteen mechanically ventilated VLBW infants with a mean corrected gestational age of 30.4 weeks participated in the study. The low frequency (LF) power slope was -17.67 (p = 0.0002) and the high frequency (HF) power slope was -0.92 (0.0003). There was a significant relationship between HF slope and birth gestational age (r = -0.49, p = 0.05). CONCLUSIONS: HF power, representing primarily parasympathetic activity, did not increase with enteral feeding as anticipated. LF power, an indicator of sympathetic tone, decreased during and after feeding suggesting the anticipated effect of inhibition of the sympathetic nervous system in response to the gut stimulus. Critically ill VLBW infants possess an overriding sympathetic response, but may not have adequate PNS tone development.

Birth Weight↗
PubMed2004

S100A2 is strongly expressed in airway basal cells, preneoplastic bronchial lesions and primary non-small cell lung carcinomas.

S100A2 gene products were shown to be frequently and dramatically over-represented in non-small cell lung cancer (NSCLC) lesions over normal tissue by microarray analysis. We have now analysed an independent series of NSCLC tumours and multiple matched normal bronchial epithelial sites by RT-PCR and immunohistochemistry to investigate: whether this expression pattern can be confirmed and whether elevated expression is associated with tumour histology, clinical outcome or preneoplasia. In this second series, S100A2 was strongly expressed in 76% (35 out of 46) of tumours, more frequently in squamous cell than adenocarcinomas (P<0.002). This strong expression was not related to high-level gene amplification, but was associated in one of five informative cases with an allele-specific imbalance in transcript levels. Most tumours strongly expressed the DeltaNp63 transcript, the product of which is a putative regulator of S100A2 transcription and while all but one of the tumours positive for DeltaNp63 expressed S100A2, others negative for this regulator also expressed the gene. Contrary to the hypothesis that S100A2 is a tumour suppressor, no somatic mutations were identified in the coding sequence in 44 tumours. Furthermore, an examination of multiple tumour-free epithelial sites from 20 patients showed that strong expression was often associated with increasing levels of disorder in preinvasive bronchial lesions (P<0.0001).

Adenocarcinoma↗
PubMed2004

Imaging of pancreatic adenocarcinoma with emphasis on multidetector CT.

Pancreatic adenocarcinoma is the fourth most frequent cause of cancer-related death. The incidence is increasing and the overall survival has altered little in recent years. Moreover, patients usually present late with inoperable disease and curative resection by standard pancreatico-duodenectomy (Whipple's procedure) is associated with significant morbidity. It should only be attempted in that small group of patients lacking radiological evidence of advanced disease. Despite the recent advances in body magnetic resonance imaging (MRI) and endoscopic ultrasound (EUS), computed tomography (CT) is the mainstay of staging in most centres and the recent development of multidetector CT machines (MDCT) has raised hope of an improvement in preoperative staging. This review focuses on the CT of pancreatic adenocarcinoma with particular emphasis on examination technique and on those criteria that determine resectability.

Adenocarcinoma↗
PubMed2003

Developing a neonatal workforce: role evolution and retention of advanced neonatal nurse practitioners.

BACKGROUND: Over the past decade more than 120 advanced neonatal nurse practitioners (ANNPs) have graduated from the Southampton based ANNP programme. OBJECTIVES: To determine the scope of practice and evolving role of ANNPs and to identify factors that may affect future recruitment and retention. METHOD: An open ended structured questionnaire on clinical role, working arrangements, retention and attrition, continuing education, and professional development was sent to 95 ANNPs. RESULTS: A response rate of 83% was achieved. There was an attrition rate of 14%, with most ANNPs remaining in their original seconding unit. Factors considered to be important for role satisfaction included a well defined role, working within a team of ANNPs, appropriate remuneration, and evidence of unit support for both role definition and continuing professional development. CONCLUSIONS: Although ANNPs are expensive to both train and employ, this evidence suggests that there is a good return on the investment in terms of retention to the unit and the specialty. Their role incorporates many features of advanced nursing practice as well as providing "value added" neonatal care by merging traditional medical and nursing roles and crossing professional boundaries. In 2004 the manpower challenges for neonatal units will be even more acute; these data confirm that there are effective options available but they require long term strategic planning and investment.

Adult↗
PubMed2002

Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival.

The most devastating aspect of cancer is the metastasis of tumour cells to organs distant from the original tumour site. The major problem facing oncologists treating uveal melanoma, the most common cancer of the eye, is metastatic disease. To lower mortality, it is necessary to increase our understanding of the molecular genetic alterations involved in this process. Using suppression subtractive hybridisation, we have analysed differential gene expression between four primary tumours from patients who have developed clinical metastasis and four primary tumours from patients with no evidence of metastasis to date. We have identified endothelin receptor type B as differentially expressed between these tumours and confirmed this observation using comparative multiplex RT-PCR. In a further 33 tumours, reduced endothelin receptor type B expression correlated with death from metastatic disease. Reduced expression also correlated with other known prognostic indicators, including the presence of epithelioid cells, chromosome 3 allelic imbalance and chromosome 8q allelic imbalance. Endothelin receptor type B expression was also reduced in four out of four primary small cell lung carcinomas compared to normal bronchial epithelium. We also show that the observed down-regulation of endothelin receptor type B in uveal melanoma was not due to gene deletion. Our findings suggest a role for endothelin receptor type B in the metastasis of uveal melanoma and, potentially, in the metastasis of other neural crest tumours.

Adult↗
PubMed2001

Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor alpha.

Estrogens play a critical role in mammary gland development, bone homeostasis, reproduction, and the pathogenesis of breast cancer by activating estrogen receptors (ERs) alpha and beta. Ligand-activated ER stimulates the expression of target proteins by interacting with specific DNA sequences: estrogen response elements (EREs). We have demonstrated that the ERE sequence and the nucleotide sequences flanking the ERE impact ERalpha binding affinity and transcriptional activation. Here, we examined whether the sequence of the ERE modulates ERalpha conformation by measuring changes in sensitivity to protease digestion. ERalpha, occupied by estradiol (E2) or 4-hydroxytamoxifen (4-OHT), was incubated with select EREs and digested by chymotrypsin followed by a Western analysis with antibodies to ERalpha. ERE binding increased the sensitivity of ERalpha to chymotrypsin digestion. We found both ligand-specific and ERE-specific differences in ERalpha sensitivity to chymotrypsin digestion. The ERE-mediated increase in ERalpha sensitivity to chymotrypsin digestion correlates with E2-stimulated transcriptional activity from the same EREs in transiently transfected cells. Transcriptional activity also correlates with the affinity of ERalpha-ERE binding in vitro. Our results support the hypothesis that the ERE sequence acts as an allosteric effector, altering ER conformation. We speculate that ERE-induced alterations in ERalpha conformation modulate interaction with co-regulatory proteins.

Allosteric Regulation↗
PubMed2001

The role of glutathione in the neurotoxicity of artemisinin derivatives in vitro.

The role of antioxidants in the neurotoxicity of the antimalarial endoperoxides artemether and dihydroartemisinin was studied in vitro by quantitative image analysis of neurite outgrowth in the neuroblastoma cell line NB2a. Intracellular glutathione concentrations were measured by high performance liquid chromatography with fluorescence detection. Both dihydroartemisinin (1 microM) and a combination of artemether (0.3 microM) plus haemin (2 microM) significantly inhibited neurite outgrowth from differentiating NB2a cells to 11.5 +/- 11.0% (SD) and 19.6 +/- 15.2% of controls, respectively. The inhibition by artemether/haemin was prevented by the antioxidants superoxide dismutase (109.7 +/- 47.8% of control), catalase (107.0 +/- 29.3%) glutathione (123.8 +/- 12.4%), L-cysteine (88.0 +/- 6.3%), N-acetyl-L-cysteine (107.8 +/- 14.9%), and ascorbic acid (104.3 +/- 12.7%). Dihydroartemisinin-induced neurotoxicity was completely or partially prevented by L-cysteine (99.5 +/- 17.7% of control), glutathione (57.9 +/- 23.4% of control), and N-acetyl-L-cysteine (57.3 +/- 9.5%), but was not prevented by superoxide dismutase, catalase, or ascorbic acid. Buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, significantly increased the neurotoxic effect of non-toxic concentrations of artemether/haemin (0.1 microM/2 microM) and dihydroartemisinin (0.2 microM), suggesting that endogenous glutathione participates in the prevention of the neurotoxicity of artemether/haemin and dihydroartemisinin. Artemether/haemin completely depleted intracellular glutathione levels, whereas dihydroartemisinin had no effect. We conclude that although glutathione status is an important determinant in the neurotoxicity of endoperoxides, depletion of glutathione is not a prerequisite for their toxicity. This is consistent with their mechanisms of toxicity being free radical-mediated damage to redox-sensitive proteins essential for neurite outgrowth, or alteration of a redox-sensitive signalling system which regulates neurite outgrowth.

Animals↗
PubMed2001

Pheochromocytoma.

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Adrenal Gland Neoplasms↗
PubMed2001

Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation.

The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.

Antigens, CD34↗
PubMed2001

Efficient expression of foreign genes in human CD34(+) hematopoietic precursor cells using electroporation.

Introduction of foreign genes into human CD34(+) hematopoietic precursor cells offers a means to correct inborn errors or to protect human stem cells from chemotherapeutic damage. Electroporation is a non-chemical, nonviral, highly reproducible means to introduce foreign genes into mammalian cells that has been used primarily for rapidly dividing cells. CD34(+) cells isolated from mobilized peripheral blood of patients were cultured for 48 h in serum-free culture medium supplemented with Flt-3 ligand, stem cell factor and thrombopoietin. Cell cycle analysis showed an increase in % S-phase from 2% on day 0 to 28% on day 2 without significant loss of mean fluorescence intensity (MFI). Optimal electroporation conditions for CD34(+) cells were 550 V/cm, 38 ms, 30 microg DNA/500 microl at cell densities between 0.2 x 10(6) and 10 x 10(6) cells/ml resulting in transient EGFP gene expression in 21% (+/- 1%) of CD34(+) precursor cells, as determined by flow cytometry 48 h after electroporation. The more primitive cells were also found to be EGFP(+) as determined by subset analysis using Thy1, CD38, AC133 and c-kit conjugated monoclonal antibodies. Methylcellulose assays on electroporated CD34(+) cells yielded 20% (+/- 7%) EGFP(+) colonies (CFU-GM, BFU-E and CFU-mix) and 22% (+/- 5%) EGFP(+) long-term colony-initiating cells (LTC-IC). The reporter gene was found to be integrated into the LTC-IC genomic DNA as determined by inverse PCR and DNA sequencing. These results suggest that electroporation has the potential to effectively and stably deliver exogenous genes into human hematopoietic precursor cells.

Antigens, CD34↗
PubMed2001

Vasorelaxant effects of oestradiols on guinea pigs: a role for gender differences.

Various studies have shown vasorelaxation properties for 17alpha- and 17beta-oestradiol. Here, we studied the effects of gender difference as well as oestrous cycle on oestradiol-induced vasorelaxation in mesenteric arteries from male and female guinea-pigs and in main uterine arteries from female guinea-pigs in vitro. Both 17alpha- and 17beta-oestradiol (0.5-20 micromol L-1) induced concentration-dependent relaxation of both mesenteric and uterine arteries preconstricted with either noradrenaline (NA; 10 micromol L-1) or KCl (125 mmol L-1) from both day-7 and day-15 female guinea-pigs. 17beta-oestradiol was more potent in relaxing arteries precontracted with NA than those pretreated with KCl, while 17alpha-oestradiol was more effective in relaxing those arteries precontracted with KCl. In mesenteric arteries from male animals, 17alpha-oestradiol was significantly (P < 0.001) more potent on arteries precontracted with NA than KCl, an effect opposite to that seen on arteries from female animals. However, both 17alpha- and 17beta-oestradiol were more potent in relaxing arteries from male animals compared with their female counterparts. These data indicate a possible role for gender differences in the vascular effects of oestradiols.

Abdomen↗