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Biomedical subjects

C A Edwards

Publications and source records attributed to C A Edwards.

At least 19 recordsLinked to original sources
PubMed2001

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

Chromosomes, Human, Pair 1↗
PubMed2001

Specific down-regulation of an engineered human cyclin D1 promoter by a novel DNA-binding ligand in intact cells.

Cyclin D1 is expressed at abnormally high levels in many cancers and has been specifically implicated in the development of breast cancer. In this report we have extensively analyzed the cyclin D1 promoter in a variety of cancer cell lines that overexpress the protein and identified two critical regulatory elements (CREs), a previously identified CRE at -52 and a novel site at -30. In vivo footprinting experiments demonstrated factors binding at both sites. We have used a novel DNA-binding ligand, GL020924, to target the site at -30 (-30-21) of the cyclin D1 promoter in MCF7 breast cancer cells. A binding site for this novel molecule was constructed by mutating 2 bp of the wild-type cyclin D1 promoter at the -30-21 site. Treatment with GL020924 specifically inhibited expression of the targeted cyclin D1 promoter construct in MCF7 cells in a concentration-dependent manner, thus validating the -30-21 site as a target for minor groove-binding ligands. In addition, this result validates our approach to regulating the expression of genes implicated in disease by targeting small DNA-binding ligands to key regulatory elements in the promoters of those genes.

Binding Sites↗
PubMed2001

Pig manure vermicompost as a component of a horticultural bedding plant medium: effects on physicochemical properties and plant growth.

This experiment was designed to characterize the physical, chemical and microbial properties of a standard commercial horticultural, greenhouse container, bedding plant medium (Metro-Mix 360), that had been substituted with a range of increasing concentrations (0%, 5%, 10%, 25%, 50% and 100% by volume) of pig manure vermicompost and to relate these properties to plant growth responses. The growth trials used tomatoes (Lycopersicon esculentum Mill.), grown in the substituted media for 31 days under glasshouse conditions, with seedling growth recorded in 20 pots for each treatment. Half of the tomato seedlings (10 pots per treatment) were watered daily with liquid inorganic fertilizer while the other half received water only. The percentage total porosity, percentage air space, pH and ammonium concentrations of the container medium all decreased significantly, after substitution of Metro-Mix 360 with equivalent amounts of pig manure vermicompost; whereas bulk density, container capacity, electrical conductivity, overall microbial activity and nitrate concentrations, all increased with increasing substitutions of vermicompost. The growth of tomato seedlings in the potting mixtures containing 100% pig manure vermicompost was reduced, possibly as a result of high soluble salt concentrations in the vermicompost and poorer porosity and aeration. The growth of tomato seedlings was greatest after substitution of Metro-Mix 360 with between 25% and 50% pig manure vermicompost, with more growth occurring in combinations of pig manure vermicompost treated regularly with a liquid fertilizer solution than in those with no fertilizer applied. Some of the growth enhancement in these mixtures seemed to be related to the combined effects of improved porosity, aeration and water retention in the medium and the high nitrate content of the substrate, which produced an increased uptake of nitrogen by the plant tissues, resulting in increased plant growth. When the tomato seedlings were watered daily with liquid inorganic fertilizer, substitution of Metro-Mix 360 with a very small amount (5%) of pig manure vermicompost resulted in a significant increase in the growth of tomato seedlings. Such effects could not be attributed solely to the nutritional or physical properties of the pig manure vermicompost. Therefore, it seems likely that the pig manure vermicompost provided other biological inputs, such as plant growth regulators into the container medium, that still need to be identified fully.

Animals↗
PubMed2000

Caveolar structure and protein sorting are maintained in NIH 3T3 cells independent of glycosphingolipid depletion.

Glycosphingolipids have been proposed to be critical components of clustered lipids within cell membranes that serve as rafts for the attachment and sorting of proteins to the cell membrane. Density gradient centrifugation was used to isolate and to ascertain the lipid composition of caveolin-enriched membranes. These membranes demonstrated a significant enrichment of sphingolipids and cholesterol containing up to 20 and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propanol, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When NIH 3T3 cells were depleted of their glucosylceramide based glycosphingolipid mass, the caveolar structure remained intact as determined by electron microscopy and confocal microscopy. The caveolar proteins caveolin and annexin II sorted normally to caveolae, as determined by immunoblotting and confocal microscopy. When the GPI-linked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depletion. These observations suggest that protein sorting to caveolae in fibroblasts occurs independently of glycosphingolipid synthesis.

3T3 Cells↗
PubMed1999

Cellular localization of a Hsp90 homologue in Porphyromonas gingivalis.

We previously reported an association between elevated serum antibody titers to the 90-kDa human heat shock protein (Hsp90), periodontal health and colonization by Porphyromonas gingivalis. In this study, we examined the cellular localization of the Hsp90 homologue of P. gingivalis. Cultures of P. gingivalis were heat-stressed (45 degrees C) and examined for localization of the Hsp90 homologue. Heat stress induced a 4-5-fold increase in anti-Hsp90 antibody reactivity over that of the unstressed controls. Western blot analysis revealed two bands (44 and 68 kDa) that reacted with anti-Hsp90 antibodies. The 68-kDa band was heat-inducible, while the 44-kDa band was not. Immunogold staining revealed that the Hsp90 homologue localized principally to the membrane and extracellular vesicles. Subcellular fractionation confirmed that the Hsp90 homologue was primarily membrane-associated.

Blotting, Western↗
PubMed1999

Hepatic apoptotic activity following transient normothermic inflow occlusion and reperfusion in the swine model.

Accelerated hepatic apoptosis was first described in portal vein-ligated livers but has since been reported in a variety of liver injuries. Because porto-prival states can induce apoptosis we sought to determine whether transient ischemic periods followed by reperfusion would trigger such cell death. The cytokines TNF-alpha and TGF-beta are known to facilitate apoptosis and are released in response to a number of stimuli including ischemia. We also investigated alterations in plasma and tissue levels of these cytokines which might lend support to their role in increased apoptotic activity following ischemia/reperfusion. Female pigs were used as the experimental model. Inflow occlusion of portal and hepatic arterial blood was performed to a portion of the swine liver directing the entire splanchnic flow to the remaining hepatic lobes for a period of 2 h. The livers were then reperfused and plasma and tissue samples taken for determination of apoptotic activity utilizing cell death immunoperoxidase staining of 3'-OH DNA ends generated by fragmentation and ELISA assay of histone-associated DNA fragments. Plasma and tissue levels of TNF-alpha and plasma levels of TGF-beta were determined by ELISA assay. An increase in apoptotic activity following reperfusion was seen at Day 2 and Day 4 compared to preischemic values by the cell death stain. The ELISA cell death assay showed an increase in apoptotic activity at 60 min, Day 2, and Day 4. Moreover, the ELISA cell death assay showed enhanced apoptotic activity in "hyperperfused" hepatic lobes compared to preischemic, or resting, liver. This was also observed when compared to sham-operated animals. Surprisingly, there was no detectable increase in plasma TNF-alpha or TGF-beta levels following ischemia/reperfusion, although homogenized liver TNF-alpha levels were increased at 60 min and Day 2 following reperfusion. We conclude that transient hepatic inflow occlusion followed by reperfusion can induce increased apoptotic activity in the swine model. Furthermore, increased apoptotic activity also occurs in the hyperperfused liver raising the possibility of a locally active factor or global hepatic expression of receptor activity in response to ischemia/reperfusion. This period of ischemia/reperfusion did not produce a detectable increase in circulating cytokine levels, and accelerated apoptosis could not be linked to heightened TNF-alpha or TGF-beta plasma activity. Higher tissue levels of TNF-alpha could reflect enhanced binding to TNF cell surface receptors or amplified receptor expression.

Animals↗
PubMed1999

Cytotoxic effects of current dental adhesive systems on immortalized odontoblast cell line MDPC-23.

OBJECTIVES: Evaluate the cytotoxic effect of the three dental adhesive systems. METHODS: The immortalized mouse odontoblast cell line (MDPC-23) was plated (30,000 cell/cm2) in 24 well dishes, allowed to grow for 72 h, and counted under inverted light microscopy. Uncured fresh adhesives were added to culture medium to simulate effects of unset adhesive. Three adhesives systems were applied for 120 min to cells in six wells for each group: Group 1) Single Bond (3M), Group 2) Prime & Bond 2.1 (Dentsply), and Group 3) Syntac Sprint (Vivadent). In the control group, PBS was added to fresh medium. The cell number was counted again and the cell morphology was assessed under SEM. In addition, the adhesive systems were applied to circles of filter paper, light-cured for 20 s, and placed in the bottom of 24 wells (six wells for each experimental materials and control group). MDPC-23 cells were plated (30,000 cell/cm2) in the wells and allowed to incubate for 72 h. The zone of inhibition around the filter papers was measured under inverted light microscopy; cell morphology was evaluated under SEM; and the MTT assay was performed for mitochondrial respiration. RESULTS: The fresh adhesives exhibited more toxic (cytopathic effects) to MDPC-23 cells than polymerized adhesives on filter papers, and as compared to the control group. The cytopathic effect of the adhesive systems occurred in the inhibition zone around the filter papers, which was confirmed by the MTT assay and statistical analysis (ANOVA) combined with Fisher's PLSD test. In the control group, MDPC-23 cells were dense on the plastic substrate and were in contact with the filter paper. In the experimental groups, when acid in the adhesive systems was removed by changing the culture medium, or when the adhesives were light-cured, some cells grew in the wells in spite of the persistent cytotoxic effect. SIGNIFICANCE: All dentin adhesive systems were cytotoxic odontoblast-like cells. Both acidity and non-acidic components of these systems were responsible for the high cytopathic effect of those dental materials.

Analysis of Variance↗
PubMed1999

Energy balance during acute respiratory exacerbations in children with cystic fibrosis.

Acute respiratory exacerbations have been proposed to contribute to the negative energy balance which causes undernutrition in cystic fibrosis. However, no studies have measured their effect on all components of energy balance. The aim of this study was to measure the effect of an acute respiratory exacerbation on energy balance. Fourteen children (six females, eight males, mean+/-SD age 9.9+/-2.4 yrs) were studied when well and during the course of an acute respiratory exacerbation treated with intravenous antimicrobial therapy. The total energy expenditure was measured using the doubly-labelled water method, resting energy expenditure by ventilated hood indirect calorimetry, energy intake by household measures records, and fat malabsorption from measurements of dietary fat intake and faecal fat output. The exacerbation was associated with a significant reduction in energy intake (mean paired difference 47 kJ x kg of body weight(-1) x day(-1), p<0.01). Changes in fat malabsorption and resting energy expenditure were negligible. The absence of significant changes in body weight and composition, together with the trend towards lower total energy expenditure, suggested no marked negative energy balance during the exacerbation. In conclusion, treatment of acute respiratory exacerbation with intravenous antimicrobial therapy represents a relatively minor challenge to energy balance and nutritional status in children with cystic fibrosis.

Acute Disease↗
PubMed1999

Adequacy of standards for assessment of growth and nutritional status in infancy and early childhood.

BACKGROUND: New "UK 1990" data have been proposed for assessing growth and nutritional status in infancy and childhood. These are still largely untried in clinical practice. There is also doubt about the applicability of more traditional reference data, which are still widely used, in assessing length, weight, skinfold thicknesses, and head circumference. AIMS: To determine the suitability of new and traditional reference data for the assessment of growth and nutritional status in infancy and early childhood. METHODS: 127 infants were recruited at birth and assessed monthly to 6 months of age then at 9, 12, 18, and 24 months. Length, weight, head circumference, and triceps and subscapular skinfold thicknesses were measured. Body mass index (BMI) was calculated. Measurements were expressed as standard deviation scores relative to "revised UK 1990" data for weight, length, head circumference and BMI; relative to Tanner-Whitehouse data for skinfold thicknesses; and relative to Gairdner-Pearson standards for head circumference. Agreement at the extremes of the distribution was assessed by comparison of observed and expected frequencies above the 90th and below the 10th centile. RESULTS: Compared with the revised UK 1990 references small differences were found for weight, length, head circumference, and BMI. Mean head circumference exceeded Gairdner-Pearson standards at all ages. Triceps and subscapular skinfold thicknesses were substantially below Tanner-Whitehouse reference data at all ages and in both sexes. CONCLUSION: Biases in revised UK 1990 reference data are small and not clinically important. The new standards are considerably more appropriate than older reference data. Use of older reference data for head circumference and skinfold thicknesses is inappropriate.

Body Height↗
PubMed1998

Probing site-specific interactions in protein-DNA complexes using heteronuclear NMR spectroscopy and molecular modeling: binding of Cro repressor to OR3.

In this paper, a general method is developed to study site-specific interactions in DNA-protein complexes using heteronuclear NMR spectroscopy and molecular modeling. This method involves two steps: (a) homonuclear 1H NMR and molecular modeling are used to develop a low resolution model and (b) 15N7-guanosine containing oligonucleotides are employed to probe the specific intermolecular interactions predicted in (a). This method is applied to Cro-operator complex due to its small size and extensive prior characterization. Non-exchangeable and exchangeable base protons have been assigned by nuclear Overhauser effect spectroscopy (NOESY) and chemical shift correlation spectroscopy. Extensive line-broadening has been observed in the 1H NMR spectra of the operator DNA in the presence of protein. Differential line-broadening observed in the imino proton region and the comparison of NOESY spectra in the presence and absence of Cro protein show that guanosine-12 and guanosine-14 are involved in the Cro-DNA interaction, while the three A.T base-pairs at the 3'- and 5'-termini play only a minor role in the binding. A model of the Cro-operator DNA complex has been constructed by docking helix-3 of the Cro protein in the major groove and it predicted specific hydrogen bonds between N7 of guanosines-12 and -14 and the side-chain of Lys-32 and Ser-28, respectively. The appearance of a new resonance in the temperature dependent proton detected heteronuclear multiple quantum coherence (HMQC) spectra of the Cro-DNA complex also demonstrates a specific interaction of Cro with guanosine-14 of the operator DNA.

DNA↗
PubMed1998

Absorption and excretion of conjugated flavonols, including quercetin-4'-O-beta-glucoside and isorhamnetin-4'-O-beta-glucoside by human volunteers after the consumption of onions.

Flavonols are polyphenols found ubiquitously in plants and plant-products. Flavonols, particularly quercetin, are potent antioxidants in vitro and their intake has been associated inversely with the incidence of coronary heart disease. The aim of this study was to investigate the accumulation in plasma and excretion in urine of flavonol glucosides following ingestion of lightly fried onions. Five healthy volunteers followed a low-flavonoid diet for 3 days. On day 4, after an overnight fast, subjects were given 300 g of lightly fried yellow onions which contain conjugates of quercetin and isorhamnetin, including quercetin-3,4 '-diO-beta-glucoside, isorhamnetin-4'-O-beta-glucoside and quercetin-4'-O-beta-glucoside. Blood collection was carried out at 0 min, 0.5, 1.0, 1.5, 2, 3, 4, 5 and 24h after the supplement. In addition, subjects collected all their urine for 24h following the onion supplement. Isorhamnetin-4'-O-beta-glucoside and quercetin-4 '-O-beta-glucoside accumulated in plasma with maximum levels, defined as proportion of intake, of 10.7+/-2.6% and 0.13+/-0.03% respectively. The time of the quercetin-4'glucoside peak plasma concentration was 1.3+/-0.2 h after the ingestion of onions while a value of 1.8+/-0.7 h was obtained for isorhamnetin-4'-glucoside. Excretion in urine, as a proportion of intake, was 17.4+/-8.3% for isorhamnetin-4'-O-beta-glucoside and 0.2+/-0.1% for quercetin-4'-O-beta-glucoside. Possible reasons for the accumulation and excretion of isorhamnetin-4'-glucoside in proportionally much higher amounts than quercetin-4'-glucoside are discussed. It is concluded that flavonols are absorbed into the bloodstream as glucosides and minor structural differences affect markedly both the level of accumulation and the extent to which the conjugates are excreted.

Adult↗
PubMed1998

Dentin-specific proteins in MDPC-23 cell line.

Only four established odontoblast-like cell lines have been reported in the literature (1-6). Of the four, only two synthesize dentin-specific proteins. These studies report that the cell line MO6-G3 synthesizes phosphophoryn (DPP), dentin sialoprotein (DSP) and dentin matrix protein-1 (DMP-1) while MDPC-23 synthesizes DSP, but not DMP-1. The objective of the present study was to determine whether polyclonal antibodies to rat DSP and DPP would label odontoblasts on microscopic sections of day-19 fetal mouse incisor odontoblasts as well as cultured cells of the MDPC-23 cell line. The spontaneously immortalized MDPC-23 cell line was derived from fetal mouse molar papillae, made continuous by the 3T6 method and cloned by dilution. These cultures have been passaged 77 times after cloning, form multilayered nodules, and have high alkaline phosphatase activity. The data show positive reactivity in odontoblasts in 19-d mouse fetal incisors as well as in cultures of MDPC-23 cells by fluorescence and confocal microscopy. In addition, these cultures were characterized by phase microscopy and scanning and transmission electron microscopy. These findings suggest that MDPC-23 cells are of the odontoblast lineage.

Animals↗
PubMed1998

Weaning practice in the Glasgow Longitudinal Infant Growth Study.

AIMS: To assess compliance with Department of Health guidelines on weaning practice in a representative sample of 127 infants from Glasgow, and to identify factors influencing timing of weaning. METHODS: Questionnaires on feeding and weaning were completed during home visits. Ninety eight mothers completed a further questionnaire on attitudes to weaning. RESULTS: Median age at introduction of solid food was 11 weeks (range 4-35 weeks); only 7% of infants had not been weaned before age 4 months. There was no difference in timing of weaning between boys and girls. Younger mothers (< 20 years old), those of lower socioeconomic status, and those who formula fed their infants tended to introduce solids earlier. Infants who were heaviest before weaning were weaned earlier. Seventy three of 98 mothers reported that they weaned their babies because they felt that they required more food. Sources of information influencing time of weaning were previous experience (53/98), books and leaflets (43/98), advice from the health visitor (31/98), and family and friends (15/98). Sixty five of 98 mothers reported receiving formal information on weaning, in most cases (54) this was from the health visitor. Mothers who received formal information tended to wean their infants later. Two per cent of infants had been given cow's milk as a main drink by age 6 months, 17% by 9 months, and 45% by the end of the first year. CONCLUSION: Compliance with recommendations on timing of weaning (not before 4 months), weaning foods, and cow's milk consumption in Glasgow is poor, although no poorer than in many other areas of the UK as found by Office of Population, Censuses and Surveys. Public health messages in relation to weaning may not be reaching their target audience.

Age Factors↗
PubMed1998

Cloned 3T6 cell line from CD-1 mouse fetal molar dental papillae.

Only primary pulpal cell cultures and one virally transformed mouse cell culture have been formally reported in the literature to synthesize proteins such as phosphophoryn which are unique to dentin matrix. In the present study, a mixed culture was derived from dental papilla cells of 18-19 fetal day CD-1 mouse mandibular first molars, maintained on a 3T6 plating regimen, and subsequently cloned after 28 passages. This cloned cell line (MDPC-23) exhibited several unique features, some of which were characteristic of odontoblasts in vivo. The features of this cell line included (1) epithelioid morphology of all cells with multiple cell membrane processes, (2) high alkaline phosphatase activity in all cells, (3) formation of multilayered nodules and multilayered cultures when maintained in ascorbic acid and beta-glycerophosphate, and (4) expression of two markers for odontoblast differentiation, i.e. dentin phosphoprotein and dentin sialoprotein.

Animals↗