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T Thomas

Publications and source records attributed to T Thomas.

At least 145 records · Page 8Linked to original sources
PubMed1998

Compensation for a gene trap mutation in the murine microtubule-associated protein 4 locus by alternative polyadenylation and alternative splicing.

One of the features expected of the gene trap approach is the functional mutation of a gene, allowing its loss-of-function phenotype analysis. We have mutated the murine microtubule-associated protein 4 (MAP-4) locus by inserting a splice-acceptor gene trap construct. Because the MAP-4 gene has been cloned, sufficient information is available to investigate the efficiency of the gene trap insertion in disrupting the protein-coding region. The fusion mRNA contains the first 905 bases of the MAP-4 mRNA and is expected to code for a truncated, nonfunctional MAP-4 protein missing, among others, the microtubule-binding domain. Activity of the lacZ marker gene of the gene trap construct was observed in all tissues throughout development and in all cells examined in adult animals. However, some cells and tissues showed higher levels of activity than others: for example, blood vessel endothelium, heart, aspects of the developing nervous system, surface ectoderm of embryonic day 11.5 embryos, and the ependymal layer and blood vessel endothelium in adult brain. MAP-4 binds to microtubules and is thought to modulate their stability. It is expressed differentially in different tissues as 5.5-kb, 6.5-kb, 8-kb, 9-kb, and 10-kb mRNA species from a single copy gene in mice. Northern hybridization with a 5', MAP-4-specific probe revealed a 3.3-kb splice variant, which has not been described previously, that was expressed as the most abundant MAP-4 mRNA species in the brain but not in other tissues. Mice homozygous for the reported gene trap insertion in the MAP-4 locus (MAP-4gt/gt) are viable and appear to be phenotypically normal. They exhibited normal levels of all MAP-4 mRNA species in brain and kidney, showing that the simian virus 40-polyadenylation signal of the gene trap construct was ignored and also showing compensation for the gene trap insertion by splicing around the gene trap construct.

Alternative Splicing↗
PubMed1998

Regulation of estrogen receptor mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin as measured by competitive RT-PCR.

Environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause alterations in gene expression. In this study, we measured the regulation of estrogen receptor (ER) mRNA in female CD-1 mice by competitive RT-PCR. Previous work suggests that ER protein levels are affected by TCDD, but how this is regulated is uncertain. These studies found no significant changes in ER mRNA levels, but the methods used (Northern blot analysis and RNase protection assays) lack sensitivity for measuring the low levels of RNA transcript, such as ER mRNA. The method described here offers an excellent alternative for quantifying the changes in mRNA levels. Internal competitors were created with gene-specific primers for ER and beta-actin by PCR reactions at low annealing temperatures. For each sample, the mRNA levels of ER and beta-actin were determined. Using competitive RT-PCR, the relative changes in ER mRNA from TCDD-treated and control animals were determined after normalization with the levels of beta-actin mRNA. The ER mRNA from female CD-1 mice treated with TCDD (single dose 5 micrograms/kg, i.p., 4 days) was found to be significantly suppressed as compared with the vehicle control in all tissues examined. TCDD decreased ER mRNA in the liver (30.1%) as expected. However, the greatest effect was in the reproductive tissues, with a 64.2% reduction in ER mRNA in the ovary. This is the first demonstration that TCDD causes tissue-specific downregulation of ER mRNA. These effects may contribute to the tissue-specific toxicity of TCDD.

Animals↗
PubMed1998

Prediction and assessment of the severity of post-operative pain and of satisfaction with management.

A prospective observational study of cohorts of patients undergoing hip replacement (30), knee replacement (31), and spinal nerve root decompressive surgery (30) were interviewed pre-operatively to identify factors which might correlate with and potentially predict severe post-operative pain and dissatisfaction with analgesic management. The hip patients comprised 33% females and averaged 64 years, while the knee patients were 45% female and older (mean 71 years) and the spinal patients were 43% female and averaged 50 years. The three groups were similar with respect to all other pre-operative variables. Pain intensity was assessed mainly by self-report using the Present Pain Intensity (PPI) and Visual Analogue Scales (VAS) of the McGill Pain Questionnaire. The PPI was preferred by patients and nurses and, as there were no analytical advantages for the VAS, the PPI data are presented. The average post-operative pain during routine management mainly with patient controlled intravenous opiate, was mild to moderate and declined over days 1-5, declined further at discharge but rose slightly 1 month after discharge. The hip replacement patients experienced significantly (P < 0.01) less pain overall than the patients in the other two groups. Nurses' assessments of pain severity from observed behaviour were low and agreed poorly with the patients' self reports. Assessed on Likert Scales (0-6), the patients generally indicated good or excellent pain control, better than expected pain experience, and high levels of satisfaction with analgesic management. Significant (P < or = 0.01) multivariate correlates of severe post-operative pain assessed by logistic regression analysis of 11 variables were female gender, high pre-operative pain severity, and younger age. Significant (P < or = 0.01) multivariate correlates of both worse than expected pain experience and low satisfaction were female gender, high pre-operative pain severity, high anxiety about risks and problems, low expected pain severity, age (younger) and high willingness to report pain. These variables may reasonably be tested in further studies as potential predictors of adverse post-operative pain experience.

Adult↗
PubMed1998

Transcriptional suppression of estrogen receptor gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

TCDD, the most potent congener of the polychlorinated dioxins, has been shown to be an antiestrogen. The mechanisms of TCDD-induced antiestrogenicity are still under investigation. In this study, we investigated the effects of TCDD on the expression of the estrogen receptor (ER) gene. We studied the levels of un-spliced ER transcript (hnRNA) as well as the ER mRNA in ovary, uterus and liver of TCDD-treated mice with different genetic backgrounds. To quantitate the ER hnRNA levels, the intron and exon boundary of ER hnRNA was amplified by competitive RT-PCR. The ER mRNA from these mice was quantitated by competitive RT-PCR amplifying exons separated by an intron. ER hnRNA and ER mRNA levels were quantitated 4 days after a single i.p. dose of TCDD (5 microg/kg) in female C57BL/6J (B6) mice, which carry the responsive allele to TCDD. TCDD treatment significantly (p < 0.05) suppressed the levels of ER hnRNA in the ovary (27.4%) and uterus (21.9%). The decreases in ER hnRNA were coordinated with significant (p < 0.01) decreases in ER mRNA in ovary (57.7%) and uterus (37.6%). There was a significant decrease (20.3%, p < 0.05) in liver ER mRNA, however, the changes of ER hnRNA in liver were not significant. The coordinated decreases in ER hnRNA and mRNA in TCDD-treated mice suggest a suppression of transcription of the ER gene. We performed the same study on DBA/2J (D2) mice, which possess the "non-responsive" allele of the aryl hydrocarbon receptor (AhR). These mice demonstrated no significant decrease in either the ER mRNA or hnRNA after TCDD treatment. Overall, these results suggest that TCDD suppresses the gene expression of the ER receptor by decreasing its transcription, and the AhR plays an important role in mediating this response.

Animals↗
PubMed1998

Induction of p21 (CIP1/WAF1/SID1) by estradiol in a breast epithelial cell line transfected with the recombinant estrogen receptor gene: a possible mechanism for a negative regulatory role of estradiol.

Estrogens stimulate the growth of a majority of estrogen receptor (ER)-positive breast cancer cells. In contrast, estradiol exerted a 75% inhibition of DNA synthesis in the MCF-10AE(wt5) cell line, obtained by the transfection of the ER gene into a normal breast epithelial cell line, MCF-10A. The estradiol-mediated growth inhibitory effect was reversed by ICI 164384, a pure anti-estrogen. Analysis of cell cycle by flow cytometry showed a significant increase of G1 cells by estradiol treatment compared to controls. To understand the mechanism of action of estradiol on MCF-10AE(wt5) cells, we examined the level of a cyclin dependent kinase inhibitor (CKI), p21, by Western blot analysis. Our results showed a 5- to 10-fold increase in the level of p21 in estradiol-treated MCF-10AE(wt5) cells compared to controls. ICI 164384 reversed estradiol-mediated induction of p21. Northern blot analysis of p21 mRNA indicated that estradiol stimulated its message in MCF-10AE(wt5) cells. Analysis of a panel of 6 breast cancer cell lines showed the absence of p21 protein, whereas it was present at a very low level in MCF-10A cells. Comparison of p21 in MCF-10A and MCF-10AE(wt5) cells showed an abundance of p21 in the ER-transfected cells. However, this p21 appears to be inactive in the absence of estradiol. These results suggest a p21-mediated pathway as a possible mechanism for the growth inhibitory effects of estradiol on at least a subset of ER-transfected cell lines.

Breast↗
PubMed1998

Polyamine inhibition of estrogen receptor (ER) DNA-binding and ligand-binding functions.

Polyamines are known to inhibit sequence specific DNA-binding activity of several zinc-finger transcription factors, including estrogen receptor (ER) binding to its cognate estrogen response element (ERE). The mechanism accounting for this disruption of protein-DNA interaction is unknown, although polyamine induction of DNA conformational changes has been suggested. To determine if polyamines can directly impair ER action, we compared the effects of putrescine (Putr), spermidine (Spd), and spermine (Spm) on ER DNA-binding (ER-ERE complex formation), ER ligand-binding (estradiol), ER structure (circular dichroism and sucrose gradient sedimentation), and the capacity of ER to transactivate an ERE-tk-CAT reporter in transient transfection assays. Polyamine concentrations causing 50% inhibition of ER-ERE formation (IC50 values) were found to be 1 mM for Putr, 4 mM for Spd, and 3 mM for Spm. This loss of ER DNA-binding was associated with a direct and irreversible effect on the ER DNA-binding domain (ER-DBD). Additionally, polyamines were observed to inhibit ER ligand-binding with IC50 values of 10 mM for Putr, 2 mM for Spd, and < 0.1 mM for Spm; and this correlated with a measureable change in higher-order ER structure (5S to 3.5S sedimentation) and inhibition of intracellular ER transactivation. These findings suggest that in ER-positive human breast tumors with increased polyamine (especially Spm) content, ER structure and function may be directly altered by tight-ion polyamine complexing that results in loss of ER-mediated gene regulation.

Breast↗
PubMed1998

Delayed healing of rhytidectomy flap resurfaced with CO2 laser.

Combining facial rhytidectomy with laser resurfacing, theoretically, provides the best opportunity for achieving an optimal facial rejuvenation result. Previous studies have demonstrated the pernicious effect of a deep peel on a skin flap, but the safety of treating the rhytidectomy flap with laser has not been investigated. This study was conducted to investigate the safety of using these techniques concomitantly. Sixty sites were selected on three Yucatan minipigs, a species of swine chosen because of its hairless nature and opportunity to raise a true skin flap (without the panniculus carnosus). The healing time of 20 laser-treated sites without flap elevation was compared with that of 20 areas treated with laser following flap elevation, shortening (to emulate a more realistic rhytidectomy process), and repair. Twenty flaps were elevated and shortened without laser treatment to serve as a control. The CO2 laser parameters were set at 500 mJ, 50 watts, and a density of 5. Two passes were made to penetrate the upper dermis. The mean healing time for areas treated with laser alone was 12.05 days, ranging from 11 to 14 days. In comparison, the healing time for the laser-treated areas subsequent to flap elevation averaged 17.95 days, with a range of 14 to 24 days (p < 0.05). Two flaps treated with laser (10 percent) failed to heal completely in 24 days. At the time that all 20 of the areas treated solely with laser had re-epithelialized completely, only one of the flaps treated with laser had re-epithelialized completely (p < 0.001). A delay in healing, as well as return of pigment, was demonstrated in the distal portions of all flaps receiving laser treatment. The control flaps all healed normally except for a 5-percent superficial loss on a single flap. It was concluded from this study, and from clinical observation of delayed healing on six of seven patients who underwent concomitant rhytidectomy and laser resurfacing at a conservative laser setting, that laser resurfacing of the rhytidectomy flap is unsafe and results in delayed re-epithelialization. This combination should be avoided altogether or performed with extreme prudence on patients undergoing a deeper plane facial rhytidectomy or by using very low laser settings.

Animals↗
PubMed1998

Systematic review of the treatment of upper respiratory tract infection.

OBJECTIVES: To assess the risks and benefits of antibiotic treatment in children with symptoms of upper respiratory tract infection (URTI). DESIGN: Quantitative systematic review of randomised trials that compare antibiotic treatment with placebo. DATA SOURCES: Twelve trials retrieved from a systematic search (electronic databases, contact with authors, contact with drug manufacturers, reference lists); no restriction on language. MAIN OUTCOME MEASURES: The proportion of children in whom the clinical outcome was worse or unchanged; the proportion of children who suffered complications or progression of illness; the proportion of children who had side effects. RESULTS: 1699 children were randomised in six trials that contributed to the meta-analysis. Six trials were not used in the meta-analysis because of different outcomes or incomplete data. Clinical outcome was not improved by antibiotic treatment (relative risk 1.01, 95% confidence interval (CI) 0.90 to 1.13), neither was the proportion of children suffering from complications or progression of illness (relative risk 0.71, 95% CI 0.45 to 1.12). Complications from URTI in the five trials that reported this outcome was low (range 2-15%). Antibiotic treatment was not associated with an increase in side effects compared with placebo (relative risk 0.8, 95% CI 0.54 to 1.21). CONCLUSIONS: In view of the lack of efficacy and low complication rates, antibiotic treatment of children with URTI is not supported by current evidence from randomised trials.

Anti-Bacterial Agents↗
PubMed1998

A signaling cascade involving endothelin-1, dHAND and msx1 regulates development of neural-crest-derived branchial arch mesenchyme.

Numerous human syndromes are the result of abnormal cranial neural crest development. One group of such defects, referred to as CATCH-22 (cardiac defects, abnormal facies, thymic hypoplasia, cleft palate, hypocalcemia, associated with chromosome 22 microdeletion) syndrome, exhibit craniofacial and cardiac defects resulting from abnormal development of the third and fourth neural crest-derived branchial arches and branchial arch arteries. Mice harboring a null mutation of the endothelin-1 gene (Edn1), which is expressed in the epithelial layer of the branchial arches and encodes for the endothelin-1 (ET-1) signaling peptide, have a phenotype similar to CATCH-22 syndrome with aortic arch defects and craniofacial abnormalities. Here we show that the basic helix-loop-helix transcription factor, dHAND, is expressed in the mesenchyme underlying the branchial arch epithelium. Further, dHAND and the related gene, eHAND, are downregulated in the branchial and aortic arches of Edn1-null embryos. In mice homozygous null for the dHAND gene, the first and second arches are hypoplastic secondary to programmed cell death and the third and fourth arches fail to form. Molecular analysis revealed that most markers of the neural-crest-derived components of the branchial arch are expressed in dHAND-null embryos, suggesting normal migration of neural crest cells. However, expression of the homeobox gene, Msx1, was undetectable in the mesenchyme of dHAND-null branchial arches but unaffected in the limb bud, consistent with the separable regulatory elements of Msx1 previously described. Together, these data suggest a model in which epithelial secretion of ET-1 stimulates mesenchymal expression of dHAND, which regulates Msx1 expression in the growing, distal branchial arch. Complete disruption of this molecular pathway results in growth failure of the branchial arches from apoptosis, while partial disruption leads to defects of branchial arch derivatives, similar to those seen in CATCH-22 syndrome.

Animals↗
PubMed1998

Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma.

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34 degrees C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5 degrees C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5 degrees C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10(-8) M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10(-8) M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5 degrees C with ascorbate and beta1-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.

Adipocytes↗
PubMed1998

A partial matching theory of the mirror effect in immediate probed recognition.

A set of experiments on immediate probed recognition of digit triples is reported in which the variables were list length (five, six, seven, or eight triples), the probability that a probe was old (.33, .5, or .67), and whether the digit triples were presented with an auditory component or articulatory suppression. Previous work had suggested that the false alarm (FA) rate in this paradigm was lower when auditory information was available than when it was not; this observation had led to the development of the partial matching theory of immediate probed recognition, according to which FAs could arise not only as a result of unlucky guesses but also when new probes shared a first digit in common with a partially retained target triple. It was argued that partial memory representations were less likely following auditory presentation than following articulatory suppression. Partial matching theory is contrasted with the rational response theory, according to which all FAs are unlucky guesses; partial matching theory gave a better account of the present experimental data than did rational response theory. However, a logical relationship between the two theories was suggested, a consequence of which was that rational response theory could be modified to include partial matching in such a way as to account for mirror effects, not only in unusually difficult immediate probed recognition tasks, but also in the more commonly studied mixed test list paradigm involving words of high or low frequency.

Adult↗
PubMed1998

The content and methodology of research papers published in three United Kingdom primary care journals.

BACKGROUND: With the expansion of academic departments, the National Research and Development initiative, and the Culyer report, United Kingdom (UK) general practice research is undergoing a period of investment and change. AIM: To examine the content and methodological quality of UK-published general practice research, and in particular to focus on the quantity and proportion of studies that were of high methodological quality, namely randomized controlled trials (RCTs). METHOD: We manually searched three UK-published journals over a five-year period: the British Journal of General Practice (BJGP), Family Practice, and the British Medical Journal (BMJ), which has a section devoted to general practice research. Studies were classified according to the International Classification of Health Problems of Primary Health Care (ICHPPC-2). RESULTS: Nearly half of published studies in UK primary care journals were concerned with either organization and administration issues in primary care or social problems (509 studies, 48%). Just over half were either qualitative studies or surveys of opinion or attitudes (528 studies, 50%). The overall number of RCTs was low (67 studies, 6%), and the proportion published has not changed over time (chi 2 for trend = 3.79, df = 1, P = 0.051). In contrast to surgical journals, nearly one-fifth of studies in general practice followed a longitudinal design (186 studies, 18%). CONCLUSIONS: The content and design of published general practice research in the UK is varied and broad. The most robust methodological design should be the aim of all prospective researchers in general practice.

Humans↗
PubMed1997

beta-Amyloid-induced cerebrovascular endothelial dysfunction.

Cerebrovascular effects of beta-amyloid were investigated using bovine mid-cerebral arteries. beta-amyloid-induced endothelial damage was evidenced by increased vasoconstriction, diminished vasodilation and was evident on electron microscopy. The endothelial dysfunction was mediated by reactive oxygen radicals. Vascular damage by beta-amyloid may be an early event in the development of the pathology of Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1997

The discharge process for palliative care patients.

This article describes the response of staff at a specialist palliative care service to the need for effective discharge planning. Hospice and community nurses were invited to give their views on the discharge of palliative care patients. The authors discuss the strategy for change and how it was implemented and evaluated.

Hospice Care↗
PubMed1997

Effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide with the estrogen response element.

We studied the effects of natural and synthetic polyamines on the conformation of an oligodeoxyribonucleotide (ODN1) harboring the estrogen response element (ERE) by circular dichroism (CD) spectroscopy and polyacrylamide gel electrophoresis. Putrescine and spermidine had no marked effect on the CD spectrum of ODN1. In contrast, spermine provoked and stabilized two characteristic changes in the CD spectrum. The first change was indicated by an increase in the intensity of the CD band at 280 nm at 0.5 mM spermine in Tris-HCl buffer containing 50 mM NaCl. This change appears to be related to changes in base tilt and conformational alterations similar to A-DNA. At 1-2 mM spermine, the CD spectrum was characterized by a loss of positive bands at 220 and 270 nm. This change might have contributions from polyamine-induced condensation/aggregation of DNA. Spectral measurements were also conducted in Tris-HCl buffer containing 150 mM NaCl to minimize contributions from condensation and aggregation of ODN1. Under these conditions, CD spectral changes were retained by (ODN1), although the magnitude of the change was diminished. In contrast, a control oligdeoxyribonucleotide (ODN2) having similar base composition did not show any significant change in the CD spectrum in the presence of 150 mM NaCl and 2 mM spermine. The changes in the CD spectrum of ODN1 were highly sensitive to polyamine structure, as evidenced by experiments using spermine analogs with altered number of -CH2- groups separating the amino and imino groups. Electrophoretic mobility shift analysis further showed ODN1 stabilization by spermine and its analogs. These data demonstrate the ability of an ODN containing ERE to undergo conformational transitions in the presence of polyamines and suggest a possible mechanism for polyamine-mediated alterations in the interaction of estrogen receptor with ERE.

Base Sequence↗