Biomedical subjects

R Smith

Publications and source records attributed to R Smith.

At least 253 records · Page 14Linked to original sources

PubMed2000

Osteoclast differentiation from circulating mononuclear precursors in Paget's disease is hypersensitive to 1,25-dihydroxyvitamin D(3) and RANKL.

A characteristic feature of Paget's disease is an increase in the number of osteoclasts in bone. Osteoclasts are formed from mononuclear phagocyte precursors that circulate in the monocyte fraction of peripheral blood. These cells require the presence of RANK ligand (RANKL)-expressing osteoblastic cells and human macrophage colony-stimulating factor (M-CSF) to form osteoclasts in vitro. To determine the role of osteoclast differentiation from circulating precursors in Paget's disease, we cultured monocytes from Paget's patients and gender- and age-matched normal controls with no evidence of bone disease for up to 21 days in the presence of UMR 106 cells and various concentrations of M-CSF (1-25 ng/mL) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] (10(-10) to 10(-7) mol/L). Relative to controls, there was a significant increase in the extent of osteoclast differentiation from pagetic monocytes as assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and lacunar bone resorption. Serial dilution experiments (2 x 10(5) to 2 x 10(2) cells/well) showed no difference in the concentration of osteoclast precursors in the peripheral blood. In Paget's patients with high serum alkaline phosphatase (sAP) levels, increased sensitivity to the osteoclastogenic effect of 1,25(OH)(2)D(3) was noted. Osteoclast differentiation did not occur when M-CSF was substituted by interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R), and these factors did not stimulate osteoclast differentiation in the presence of M-CSF. In this in vitro coculture system, osteoclast formation was inhibited by osteoprotegerin in a dose-dependent manner. In the presence of RANKL (5-30 ng/mL) and M-CSF (25 ng/mL), osteoclast formation and bone resorption were significantly increased in cultures of monocytes from patients with high and low sAP levels as compared with normal controls. Our findings suggest that the increase in osteoclast numbers seen in Paget's disease results not from an increase in the number of circulating precursors in peripheral blood but rather from an increased sensitivity of osteoclast precursors to the humoral factors, 1,25(OH)(2)D(3) and RANKL, which regulate osteoclast formation.

Acid Phosphatase↗

PubMed2000

The embodiment of value: C.S. Sherrington and the cultivation of science.

The paper examines the ruputation of C.S. Sherrington as both eminent physiologist and eminent representative of scientific culture. It describes Sherrington's 'figurehead' status. In his career, research and personal manner, he embodied a life of science, not only in opposition to humanistic values but in fact appearing to be the highest achievement of those values. An analysis of Sherrington's research, of his lectures on Man on His Nature and of his poetry supports this account. The paper uses Sherrington's reputation to describe the values of an establishment group of English-speaking scientists and physicians in the 1930s and 1940s.

England↗

PubMed2000

The role of outbreaks in developing food safety policy: population based surveillance of salmonella outbreaks in Wales 1986-98.

In developing public policy on food safety, systematic identification and thorough investigation of all general outbreaks is necessary in order to avoid bias towards highly publicised outbreaks. In Wales, from 1986 to 1998, 87 general foodborne outbreaks of salmonellosis were identified. Most outbreaks occurred at functions or were associated with small catering outlets such as bakeries and sandwich bars. In 50 outbreaks, a vehicle of infection was confirmed microbiologically and/or epidemiologically. The most common food vehicles were those containing shell eggs. Salmonella enteritidis outbreaks were significantly more likely than outbreaks of other serotypes to be associated with vehicles containing shell eggs, suggesting that eggs were also the source of infection in many outbreaks. The routine use of analytical epidemiological studies to identify vehicles in outbreaks is recommended.

Disease Outbreaks↗

PubMed2000

A test of the model to predict unusually stable RNA hairpin loop stability.

To investigate the accuracy of a model [Giese et al., 1998, Biochemistry37:1094-1100 and Mathews et al., 1999, JMol Biol 288:911-940] that predicts the stability of RNA hairpin loops, optical melting studies were conducted on sets of hairpins previously determined to have unusually stable thermodynamic parameters. Included were the tetraloops GNRA and UNCG (where N is any nucleotide and R is a purine), hexaloops with UU first mismatches, and the hairpin loop of the iron responsive element, CAGUGC. The experimental values for the GNRA loops are in excellent agreement (deltaG degrees 37 within 0.2 kcal/mol and melting temperature (TM) within 4 degrees C) with the values predicted by the model. When the UNCG hairpin loops are treated as tetraloops, and a bonus of 0.8 kcal/mol included in the prediction to account for the extra stable first mismatch (UG), the measured and predicted values are also in good agreement (deltaG degrees 37 within 0.7 kcal/mol and TM within 3 degrees C). Six hairpins with unusually stable UU first mismatches also gave good agreement with the predictions (deltaG degrees 37 within 0.5 kcal/mol and TM within 8 degrees C), except for hairpins closed by wobble base pairs. For these hairpins, exclusion of the additional stabilization term for UU first mismatches improved the prediction (AG degrees 37 within 0.1 kcal/mol and TM within 3 degrees C). Hairpins with the iron-responsive element loop were not predicted well by the model, as measured deltaG degrees 37 values were at least 1 kcal/mol greater than predicted.

Base Pair Mismatch↗

PubMed2000

Dynamic mechanical characterization of hydroxyapatite reinforced polyethylene: effect of particle size.

Dynamic mechanical analysis (DMA) was used to characterize biomedical composites consisting of synthetic hydroxyapatite (HA) particulate reinforced polyethylene (PE). The effects of the HA volume fraction, temperature and HA particle on the storage modulus (E(I)) and damping (tan delta) were investigated. Increasing HA volume fractions increased E(I) and decreased tan delta. E(I) was found to be linearly related to the Young's modulus values obtained from quasi-static tensile tests. Relative modulus and damping studies showed that the viscoelastic behavior of unfilled PE was different to that of the filled matrix due to the presence of thermally induced tensile stresses in the matrix at the filler-matrix interface.

Journal Article↗

PubMed2000

Linkage exclusion and mutational analysis of the noggin gene in patients with fibrodysplasia ossificans progressiva (FOP).

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder characterized by congenital malformation of the great toes and by progressive heterotopic endochondral ossification in predictable anatomical patterns. Although elevated levels of bone morphogenetic protein 4 (BMP4) occur in lymphoblastoid cells and in lesional cells of patients with FOP, mutations have not been identified in the BMP4 gene, suggesting that the mutation in FOP may reside in a BMP4-interacting factor or in another component of the BMP4 pathway. A powerful antagonist of BMP4 is the secreted polypeptide noggin. A recent case report described a heterozygous 42-bp deletion in the protein-coding region of the noggin gene in a patient with FOP. In order to determine if noggin mutations are a widespread finding in FOP, we examined 31 families with 1 or more FOP patients. Linkage analysis with an array of highly polymorphic microsatellite markers closely linked to the noggin gene was performed in four classically-affected multigenerational FOP families and excluded linkage of the noggin locus to FOP (the multipoint lod score was -2 or less throughout the entire range of markers). We sequenced the noggin gene in affected members of all four families, as well as in 18 patients with sporadic FOP, and failed to detect any mutations. Single-strand conformation polymorphism (SSCP) analysis of 4 of these patients plus an additional 9 patients also failed to reveal any mutations. Among the samples analyzed by SSCP and DNA sequencing was an independently obtained DNA sample from the identical FOP patient previously described with the 42-bp noggin deletion; no mutation was detected. Examination of the DNA sequences of 20 cloned noggin PCR products, undertaken to evaluate the possibility of a somatic mutation in the noggin gene which could be carried by a small subset of white blood cells, also failed to detect the presence of the reported 42-bp deletion. We conclude that mutations in the coding region of noggin are not associated with FOP.

Carrier Proteins↗

PubMed2000

Mouse mutants from chemically mutagenized embryonic stem cells.

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

Abnormalities, Drug-Induced↗

PubMed2000

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge.

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

Amino Acid Motifs↗

PubMed2000

Efficient and sustained transgene expression in human corneal cells mediated by a lentiviral vector.

The development of vectors and techniques able to transfer potentially therapeutic genetic information to corneal tissues efficiently may have broad clinical applications. Although a variety of vectors have been tested for their ability to transduce corneal tissue, these systems have been ineffective at transducing all cell types or have been associated with a relatively short duration of transgene expression. Towards the implementation of efficient, long-term transgene expression in all corneal cell types, we have studied the ability of a recombinant lentiviral vector, containing the enhanced green fluorescent protein (eGFP), to mediate gene transfer into human corneal tissue in vitro and in situ. Human primary keratocytes, cultured in vitro, were efficiently transduced by a lentiviral vector as determined by fluorescent-activated cell sorting (FACS) and by fluorescent microscopy. Transduction efficiency was found to be dependent upon multiplicity of infection (MOI); 92% of keratocytes were transduced at an MOI of 1000. The proportion of eGFP-positive cells remained unchanged throughout continuous culture for 60 days, indicating stable expression and a lack of selective pressure for or against transduced cells. Human corneal tissue, obtained at the time of penetrating keratoplasty, demonstrated efficient in situ transduction with this vector. Endothelial cells, epithelial cells and stromal keratocytes at the exposed cut edge of the corneal tissue in situ demonstrated eGFP expression. Underlying stromal cells not in direct contact with vector-containing media, were not transduced, implying that virus-cell contact is required for transduction. Transduced corneal tissues expressed eGFP in situ for the life of the corneal button in extended organ culture (60 days). These results imply that lentiviral vectors may prove to be useful tools, able to transduce corneal tissue efficiently, and that transgene expression is temporally stable. Gene Therapy (2000) 7, 196-200.

Cells, Cultured↗

PubMed2000

Selecting donors of platelets for refractory patients on the basis of HLA antibody specificity.

BACKGROUND: Patients who are refractory to platelet transfusion as a result of HLA alloimmunization are generally given HLA-matched or crossmatched platelets. However, HLA-matched platelets that are matched at HLA-A and -B loci (A-matched) or those without any mismatched or cross-reactive antigens (BU-matched) are frequently unavailable. A disadvantage of crossmatching is that crossmatched platelets have a shelf life of only 5 days, so that crossmatch tests must be performed frequently for patients requiring long-term platelet transfusions. An alternative method is the selection of platelets according to the patient's HLA antibody specificity, called the antibody specificity prediction (ASP) method. STUDY DESIGN AND METHODS: An anti-human globulin-enhanced microlymphocytotoxicity test modified by a double addition of serum and a computer program were used to determine the specificity of patients' HLA antibodies. Platelet crossmatching was performed with a solid-phase adherence assay. The percentage of platelet recovery (PPR) was determined in 1621 platelet transfusions in an observational study in 114 patients, and the PPR of platelets selected by the ASP method was compared with the PPR of those that were HLA-matched, crossmatched, or randomly selected. The numbers of potential donors in files of HLA-typed donors as identified by HLA matching vs. the ASP method were determined. RESULTS: After adjustments for covariates, the mean +/- SEM PPR was similar for HLA-matched (21 +/-4%), cross-matched (23+/-4%), and ASP-selected (24+/-3%) platelets and was significantly lower for randomly selected (15+/-1.4%) platelets. For 29 alloimmunized HLA-typed patients, the mean number of potential donors found in a file of 7247 HLA-typed donors was 6 who were an HLA-A match (median = 1), 33 who were an HLA-BU match (median = 20), and 1426 who were identified by the ASP method (median = 1365). CONCLUSION: The ASP method of donor selection for refractory alloimmunized patients appears as effective as HLA matching or crossmatching. Far more donors are identified in a file of HLA-typed donors by the ASP method than by HLA matching, and this indicates that the ASP method provides important advantages regarding the availability of compatible platelet components.

Analysis of Variance↗

PubMed2000

Prevalence of HIV among pregnant women in Dundee 1988-1997: evidence to gauge the effectiveness of HIV prevention measures.

OBJECTIVES: During November 1988-July 1990, an HIV prevalence survey of pregnant women in Dundee, which used a combined voluntary diagnostic testing and voluntary unlinked anonymous approach, revealed a rate of 0.3%, the highest recorded prevalence among such a population in the U.K. at the time. To determine if, and why, any changes in HIV prevalence had occurred during the early to mid 1990s, further studies were conducted. METHODS: During January 1993-December 1997, antenatal patients of, and women undergoing therapeutic termination of pregnancy at, Ninewells Hospital, Dundee, were offered a diagnostic HIV antibody test. For those declining, residual sera from rubella specimens were tested for HIV antibodies using an unlinked anonymous approach which did not necessitate the securement of informed consent. Information about injecting drug use was obtained from all women and linked to their HIV test results. RESULTS: For all pregnant women, a significant decline in HIV prevalence (P<0.05) from 0.3% (19/6228) during 1988-1990 to 0.12% (22/17899) during 1993-1997 was observed. For those who injected drugs, prevalence decreased significantly (P<0.05) from 27.5% (11/40) to 7% (6/85), while among women who had never injected drugs prevalence decreased slightly, but not significantly (P>0.05), from 0.13% (8/6188) to 0.09% (16/17814).Prevalence in the non-injectors who reported no sexual intercourse with an injector was low at 0.04% (8/17682) during 1993-1997. No significant trends in HIV prevalence were seen over the 5 years up to 1997. CONCLUSION: The decline in HIV prevalence among pregnant women in Dundee during the early to mid-1990s can be explained predominantly by the control of HIV transmission among the city's injectors, and from them to the wider heterosexual population. It is likely that interventions designed to reduce needle/syringe sharing among injectors have been successful. It is imperative that the preventive effort is not allowed to be weakened.

Abortion, Induced↗

PubMed2000

Fetal placental vascular responses to corticotropin-releasing hormone in vitro. Effects of variation in oxygen tension.

In this study, using the human placenta perfused in vitro with Krebs' bicarbonate solution, we have examined the effects of changes in oxygen tension on the vasoreactivity of fetal placental blood vessels to corticotropin releasing hormone (CRH). Vasodilatory responses to human synthetic CRH were measured during sub-maximal vasoconstriction of the fetal placental circulation with prostaglandin F(2alpha)(PGF(2alpha)) (1-100 micrometer). Decreases in fetal placental arterial perfusion pressure (FAP) were obtained with CRH under conditions of high oxygen or low oxygen tension, >/=450 mmHg and </=50 mmHg, respectively. Secretion of CRH into the maternal and fetal placental circulations was measured during changes in oxygen tension in normal placentae and placentae from abnormal pregnancies complicated by pre-eclampsia. The change from high to low oxygen perfusion resulted in a small increase in the basal perfusion pressure (21+/-3.6 to 28.3+/-2.6 mmHg; (P</= 0.001, Student's paired t -test). During high oxygen perfusion, CRH (0. 3-3000 p m) caused a concentration dependent reduction of the PGF(2alpha)induced increase in FAP. However, during low oxygen perfusion, the vasodilatory effects of CRH were completely inhibited (P</= 0.05, regression analysis, ANOVA). The effect of the NO synthase inhibitor l -nitro-omega-arginine methyl ester (l -NAME, 1-100 micrometer), on basal FAP during high and low oxygen conditions was also established. Low oxygen perfusion significantly attenuated l -NAME-induced increases in perfusion pressure (P</= 0.05, regression analysis, ANOVA). Low oxygen perfusion was associated with an increase in CRH secretion into the maternal but not fetal circulation. CRH release into either the maternal or fetal circulations of abnormal placentae were not significantly different from normal controls. In conclusion CRH-induced vasodilatation of the fetal placental vasculature in vitro is inhibited during low oxygen perfusion. This effect may be related to reduced NO production. Reduced CRH induced vasodilation is associated with increased secretion of the CRH into the maternal but not fetal circulation.

Adult↗

PubMed2000

Localization and characterization of urocortin during human pregnancy.

Urocortin, a recently identified peptide of the corticotropin releasing hormone (CRH) peptide family, has potent vasodilatory effects in the human fetal placental circulation in vitro, promoting us to hypothesize that urocortin is produced locally to regulate uteroplacental vascular tone during pregnancy. In the present study, we examined the distribution of urocortin in the human placenta, fetal membranes and uterine tissue at term in the presence and absence of labour, using a urocortin antibody produced in our laboratory and the immunoperoxidase staining method. Immunoreactive (IR)-urocortin was observed in the vascular smooth muscle of the myometrium (n=5), decidual stromal cells, syncytiotrophoblast and amnion epithelium (n=10). No differences in staining intensity for urocortin were detected between tissues obtained in the absence (n=5) or presence (n=5) of labour. Staining intensity for IR-urocortin was greatest in the decidua suggesting this may be a site of urocortin production during pregnancy. Subsequently, we tested urocortin secretion from chorio-decidual cells in vitro, using an immunoblot technique. Positive staining for urocortin was observed in 40 per cent of chorio-decidual cells with 34 per cent of these cells secreting urocortin under basal conditions. Since urocortin was secreted by decidual cells we questioned whether urocortin was present in maternal plasma throughout gestation, using radioimmunoassay. Urocortin was detectable in maternal plasma from 7 weeks of gestation and concentrations did not change as gestation progressed. IR-urocortin in the maternal plasma eluted from a Sephadex G-50 column at the same site as synthetic urocortin and had a calculated retention coefficient (Kd) of 0.44. In summary, this study indicates that urocortin is produced by the decidua during human pregnancy and is detectable in maternal plasma. These data are consistent with the hypothesis that urocortin is produced locally by the decidua and may act to regulate uteroplacental blood flow.

Amnion↗

PubMed2000

Differences in the concentration of various synovial fluid constituents between the distal interphalangeal joint, the metacarpophalangeal joint and the navicular bursa in normal horses.

As a prerequisite for the identification of navicular disease markers, the concentrations of cartilage oligomeric matrix protein (COMP), total glycosaminoglycans (GAG), hyaluronan, metalloproteinases (MMP) 2 and 9 and total protein were measured in synovial fluid samples obtained from the distal interphalangeal joint (DIP), the metacarpophalangeal joint (MCP) and the navicular bursa of 24 horses. Mean GAG, COMP and total protein levels were significantly higher in the DIP joint and in the navicular bursa compared to the MCP joint. Hyaluronan content was lower. MMP -2 activity was present in all fluids measured and had similar levels in different joints. MMP -9 was present in 42 per cent of MCP joint samples and 58 per cent of DIP joint samples and of navicular bursal samples. In relation to the constituents measured, the composition of navicular bursal fluid was similar to the articular synovial fluids, in particular that obtained from the DIP joint. Correlation between the constituents of DIP joint fluid and navicular bursal fluid obtained from the same legs was statistically significant for all the parameters measured.

Animals↗

PubMed2000

Fibrodysplasia ossificans progressiva, a heritable disorder of severe heterotopic ossification, maps to human chromosome 4q27-31.

Fibrodysplasia ossificans progressiva (FOP) is a severely disabling, autosomal-dominant disorder of connective tissue and is characterized by postnatal progressive heterotopic ossification of muscle, tendon, ligament, and fascia and by congenital malformation of the great toes. To identify the chromosomal location of the FOP gene, we conducted a genomewide linkage analysis, using four affected families with a total of 14 informative meioses. Male-to-male transmission of the FOP phenotype excluded X-linked inheritance. Highly polymorphic microsatellite markers covering all human autosomes were amplified by use of PCR. The FOP phenotype is linked to markers located in the 4q27-31 region (LOD score 3.10 at recombination fraction 0). Crossover events localize the putative FOP gene within a 36-cM interval bordered proximally by D4S1625 and distally by D4S2417. This interval contains at least one gene involved in the bone morphogenetic protein-signaling pathway.

Chromosome Mapping↗

PubMed2000

Correlation of CD34+ cell counts with volume of bone marrow collected for allogeneic bone marrow harvests.

The quantity of bone marrow collected for allogeneic bone marrow transplantation is based on collecting 10 to 15 cc of bone marrow/kg of recipient weight. We hypothesized that the percentage of CD34+ cells collected during a bone marrow harvest decreased at the end of the harvest because of increasing amounts of peripheral blood contamination. We performed a prospective, blinded study in which we measured CD34+ percentages and cell counts at 200-cc intervals during bone marrow harvests from 11 consecutive human leukocyte antigen (HLA)-matched sibling bone marrow donors. We observed that the percentage of CD34+ cells in aspirated bone marrow did not vary significantly from the start to the end of the bone marrow harvest, and the total number of CD34+ cells/kg increased in a linear fashion, thus disproving our original hypothesis. In conclusion, the percentage of CD34+ cells in aspirated bone marrow will remain constant throughout a bone marrow harvest.

Adolescent↗

PubMed2000

Analysis of mid-trimester corticotrophin-releasing hormone and alpha-fetoprotein concentrations for predicting pre-eclampsia.

The aims of this study were firstly to examine if corticotrophin-releasing hormone (CRH) concentrations in maternal plasma were significantly elevated in Chinese pregnancies complicated by pre-eclampsia, secondly to assess if this elevation could be detected in the mid-trimester before onset of clinical signs of the disease, and thirdly to evaluate the performance of using maternal CRH and/or alpha-fetoprotein (AFP) concentrations in the mid-trimester for prediction of pre-eclampsia. The first part of this study was tested in a cohort of 39 subjects. The CRH concentrations were significantly elevated in pregnant women complicated by pre-eclampsia. The second and third parts of the study involved a different cohort of 1021 subjects. Both CRH and AFP concentrations in the mid-trimester were significantly elevated in those who subsequently developed pre-eclampsia. However, when used for prediction of pre-eclampsia, neither the CRH nor AFP concentrations alone in the mid-trimester had strong predictive value. Although the combination of both tests improved the detection rate compared to the use of CRH alone, the small increase in the likelihood ratio from 1.9 to 2.6 did not suggest that the combination would be of great clinical value.

Adult↗

PubMed2000

A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity.

A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10(-9) M 17beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10(-12) M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0(-10) M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17beta-estradiol.

Cell Division↗