Biomedical subjects

W Porter

Publications and source records attributed to W Porter.

At least 37 records · Page 2Linked to original sources

PubMed1998

Estrogen-induced retinoic acid receptor alpha 1 gene expression: role of estrogen receptor-Sp1 complex.

Retinoic acid receptor alpha 1 (RAR alpha 1) gene expression is induced by 17 beta-estradiol (E2) in estrogen receptor (ER)-positive breast cancer cells, and the -100 to -49 region of the RAR alpha 1 gene promoter was previously shown to be required for E2-responsiveness. This region of the RAR alpha 1 promoter was further analyzed using the following oligonucleotides: -100 to -49 (RAR4); -79 to -56 (RAR3); -79 to -49 (RAR2); -100 to -58 (RAR1); and their derived promoter reporter constructs (pRAR4, pRAR3, pRAR2, and pRAR1). In transient transfection studies in MCF-7 human breast cancer cells, pRAR2 and pRAR1 were E2-responsive; both of the RAR alpha 1 gene promoter inserts contained two GC-rich sites and bound Sp1 protein in gel mobility shift assays. Using wild-type [32P]RAR2 and oligonucleotides mutated in one or both GC-rich sites, it was shown that ER enhanced Sp1 binding to both sites, but a ternary ER-Sp1-DNA complex was not observed in gel mobility shift assays. In transient transfection assays, each of the GC-rich motifs were sufficient for E2-induced transactivation. In ER-negative MDA-MB-231 cells transiently transfected with pRAR2, E2 responsiveness was observed only in cells cotransfected with wild-type ER or 11C-ER containing a deletion of the DNA-binding domain but not with ER variants that express activation function-1 (AF-1) or AF-2. Using a similar approach, it was shown that the GC-rich sites in RAR1 were also sufficient for ER activation. These results demonstrate that interaction of a transcriptionally active ER/Sp1 complex with GC-rich motifs is required for hormone inducibility of the downstream region of the RAR alpha 1 gene promoter.

Adenocarcinoma↗

PubMed1997

Identification of a functional imperfect estrogen-responsive element in the 5'-promoter region of the human cathepsin D gene.

17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells. Previous studies have identified an Sp1-imperfect estrogen-responsive element (ERE) half-site [GGGCGG(N)23ACGGG] (-199 to -165) in the promoter region which forms an Sp1-estrogen receptor (ER) complex and confers E2 responsiveness on the corresponding Sp1-ERE-chloramphenicol acetyl transferase (CAT) construct. Further analysis of downstream regions of the promoter identified a CGCCC(N)3TGACC sequence (-119 to -107) which is homologous to the adenovirus major late promoter element (MLPE) and binds the ER to form a retarded band in a gel electrophoretic mobility shift assay. The corresponding promoter-CAT construct is also E2-inducible. The MLPE resembles an imperfect palindromic ERE containing imperfect (5') and perfect (3') ERE half-sites; analysis of oligonucleotides with mutations in these half-sites shows that only the perfect ERE half-site is required for binding the ER, whereas both sites are required for transactivation. In vivo exonuclease III footprinting showed that treatment with E2 also enhanced binding at the MLPE site. Identification of this second functional enhancer sequence in the 5'-promoter region of cathepsin D is consistent with the increasingly complex cell-specific regulation of hormone-responsive genes.

Base Sequence↗

PubMed1997

Identification of a motif within the 5' regulatory region of pS2 which is responsible for AP-1 binding and TCDD-mediated suppression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds modulate several endocrine systems by altering hormone synthesis, enhancing ligand metabolism, and down-regulating receptor levels/binding activity. Previous studies have demonstrated that TCDD inhibits the 17beta-estradiol (E2)-induction of pS2, a human breast cancer prognostic marker. This inhibition occurs at the gene expression level and is Ah receptor (AhR)-mediated. Analysis of the 5' regulatory region has identified three motifs which resemble dioxin response element (DRE) core sequences. pS2-regulated luciferase deletion constructs identified the DRE-like motif located at -527 to -514 as being required for TCDD-mediated suppression. A point mutation within this core motif (T-518C) abolished the inhibition by TCDD while UV-induced protein-DNA cross-linking and competitive gel retardation assays demonstrated AhR complex binding to this motif. Further study of this region also revealed an adjacent putative AP-1 site, diverging by one base pair from the consensus sequence. Gel retardation assays using TPA-treated MCF-7 cell nuclear extracts showed an induced complex binding to the AP-1-like site. Competition studies and antibody supershifts confirmed that the retarded complex consists of AP-1-like proteins. pS2-regulated luciferase constructs containing mutations specific to the AP-1-like motif greatly diminished the inducibility in response to E2. These results suggest that an interaction between AhR complexes and AP-1-like proteins may be responsible for TCDD-mediated inhibition of E2-induced pS2 expression.

Base Sequence↗

PubMed1997

Mechanism of inhibition of MDA-MB-468 breast cancer cell growth by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Treatment of estrogen receptor (ER)-negative MDA-MB-468 human breast cancer cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced formation of a nuclear aryl hydrocarbon (Ah) receptor complex as determined by ligand-binding and gel electrophoretic mobility shift assays. TCDD also induced CYP1A1-dependent activity in MDA-MB-468 cells, which represents the first ER-negative Ah receptor-positive human breast cancer cell line that has been identified. Treatment of this cell line with TCDD and related compounds also caused a 50% inhibition of cell growth, which resembled the growth inhibitory effects previously reported for epidermal growth factor (EGF). However, EGF expression is minimal in this cell line and is not induced by TCDD; moreover, EGF and TCDD induced a different pattern of oncogene expression and apoptosis in MDA-MB-468 cells. In contrast, TCDD caused a rapid and sustained induction of transforming growth factor alpha (TGF alpha) gene expression and secreted protein (nearly 2-fold); moreover, the growth-inhibitory effects of TCDD could be blocked by antibodies to the EGF receptor. In a separate experiment, it was shown that TGF alpha also inhibited growth of MDA-MB-468 cells. The results of this study indicate that the mechanism of growth inhibition of MDA-MB-468 cells by TCDD is due to induction of TGF alpha, which is a potent antimitogen in this cell breast cancer line.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed1997

Functional synergy between the transcription factor Sp1 and the estrogen receptor.

A GC-rich oligonucleotide containing an estrogen responsive element (ERE) half-site from the heat shock protein 27 (Hsp 27) gene promoter (-105 to -84) [ie. GGGCGGG(N)10GGTCA; Sp1(N)10ERE] forms a complex with the Sp1 and estrogen receptor (ER) proteins. Moreover, promoter-reporter constructs containing this sequence (-108 to -84 or -108 to +23) are also estrogen-responsive. Mutation of the ERE half-site in the Hsp 27-derived oligonucleotides did not result in loss of estrogen responsiveness in transient transfection studies, suggesting that estrogen inducibility was mediated through the Sp1-DNA motif. Gel mobility shift assays using 32P-labeled wild type and ERE mutant Sp1(N)10ERE and consensus Sp1 oligonucleotides showed that Sp1 protein formed a DNA-protein complex with all three nucleotides, and the intensities of retarded bands were enhanced by coincubation with wild type ER and 11C-ER, which does not contain the DNA-binding domain. ER mutants in which N-terminal (19C-ER) and C-terminal (15C-ER) regions were deleted did not enhance Sp1-DNA binding or hormone-induced transactivation of GC-rich promoter-reporter constructs in ER-negative MDA-MB-231 cells, whereas both wild type and 11C-ER restored inducibility. Immunoprecipitation studies also confirmed that the Sp1 and ER proteins physically interact. The interaction of the Sp1 and ER proteins and the resulting enhanced Sp1-DNA binding is observed in the presence or absence of estrogen (hormone-independent), whereas transactivation of promoter-reporter constructs is estrogen-dependent. Thus, the results illustrate a new estrogen-dependent transactivation pathway that involves ER-protein interactions and is ERE-independent.

Animals↗

PubMed1996

Effect of transient expression of the oestrogen receptor on constitutive and inducible CYP1A1 in Hs578T human breast cancer cells.

Hs578T human breast cancer cells are an oestrogen receptor (ER)-negative cell line. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in formation of a 6.9 S nuclear aryl hydrocarbon (Ah) receptor complex, which bound to a [32P]dioxin-responsive element in a gel electrophoretic mobility shift assay. However, TCDD does not induce CYP1A1 gene expression or chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with pRNH11c or pMCAT5.12, which are Ah-responsive plasmids derived from the 5'-flanking region of the human and murine CYP1A1 genes respectively. Restoration of Ah responsiveness was investigated by co-transfecting Hs578T cells with pRNH11c or pMCAT5.12 and plasmids that express the ER (hER), Ah receptor (AhR) and AhR nuclear translocator (Arnt) proteins. ER expression resulted in significantly increased basal CAT activity; however, TCDD did not induce CAT activity in the transiently transfected cells. Expression of the AhR or Arnt proteins did not alter basal or inducible CAT activity. Expression of N- or C-terminal truncated ER in Hs578T resulted in differential regulation of Ah responsiveness. In Hs578T cells transiently expressing the ER, which contains C-terminal deletions (amino acids 282-595), basal CAT activity was also increased; however, Ah responsiveness was not restored. In contrast, transient expression of N-terminal-deleted (amino acids 1-178) ER resulted in a marked decrease in basal CAT activity but a restoration of Ah responsiveness. These results suggest that basal and inducible CAT activity in Hs578T cells transiently transfected with pRNH11c is modulated differentially by ER domains that are present in the N- and C-terminal regions of the ER.

Base Sequence↗

PubMed1996

Role of estrogen receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene expression.

Treatment of MCF-7 human breast cancer cells with 10 nM 17 beta-estradiol (E2) resulted in a 2-fold induction of heat shock protein (Hsp) 27 mRNA levels, and this response persisted for up to 24 h. The 5'-promoter region of the gene was further investigated to identify genomic sequences associated with E2 responsiveness. An Sp1 and half-palindromic estrogen response element (ERE) separated by 10 nucleotides, GGGCGGG(N)10GGTCA, were identified at -105 to -84, and formation of the Sp1/estrogen receptor (ER) complex was investigated by in vitro assays using synthetic Hsp 27-[32P]Sp1/ERE oligonucleotides in a gel mobility shift assay and transient transfection studies using short (-108/-84) and long (-108/+23) 5'-promoter sequences linked to a thymidine kinase promoter and the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (Hsp-CATs and Hsp-CATl, respectively). Incubation of nuclear extracts from MCF-7 cells with an Hsp 27-[32P]Sp1/ERE oligonucleotide results in formation of an Sp1/ER complex. The formation of this complex was inhibited by coincubation with unlabeled Sp1/ERE, ERE, and Sp1 oligonucleotides and by preincubation with ER or Sp1 antibodies (immunodepletion). In addition, the complex was supershifted by coincubation with ER antibodies. Mutation of either Sp1 or ERE sites also decreases formation of the retarded band. E2 induced CAT activity in MCF-7 cells transiently transfected with either Hsp-CATs or Hsp-CATl plasmids. It was also demonstrated that E2 did not significantly induce CAT activity in MCF-7 cells transiently transfected with Hsp-CATl-containing mutations in both the Sp1 and ERE sites. The results of this study demonstrate that an Sp1/ER complex is involved in E2-induced Hsp 27 gene expression.

Antibodies↗

PubMed1995

Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells.

17 beta-Estradiol (E2) induces cathepsin D mRNA levels and intracellular levels of immunoreactive protein in MCF-7 human breast cancer cells. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alone does not affect cathepsin D gene expression in this cell line; however, in cells cotreated with TCDD and E2, TCDD inhibited E2-induced cathepsin D mRNA levels, the rate of gene transcription, and levels of immunoreactive protein. The inhibitory responses were observed within 30 to 120 min after the cells were treated with TCDD. TCDD also inhibited E2-induced secreted alkaline phosphatase activity in aryl hydrocarbon (Ah)-responsive MCF-7 and wild-type mouse Hepa 1c1c7 cells cotransfected with the human estrogen receptor (hER) and the pBC12/S1/pac plasmid, which contains the 5' promoter region (-296/+57) of the cathepsin D gene and an alkaline phosphatase reporter gene. The E2-responsive ER/Sp1 sequence (-199 to -165) in the cathepsin D 5' region contains an imperfect GTGCGTG (-175/-181) xenobiotic responsive element (XRE); the role of this sequence in Ah responsiveness was investigated in gel electrophoretic mobility shift assays and with plasmid constructs containing a wild-type ER/Sp1 oligonucleotide or a mutant ER/Sp1-"XRE" oligonucleotide containing two C-->A mutations in the XRE sequence (antisense strand). In plasmid constructs which contained a chloramphenicol acetyltransferase reporter gene and the wild-type ER/Sp1 promoter sequence, E2-induced chloramphenicol acetyltransferase activity and mRNA levels were inhibited by TCDD whereas no inhibition was observed with the mutant ER/Sp1-"XRE" plasmids. Electrophoretic mobility shift assays showed that the nuclear or transformed cytosolic Ah receptor complex blocked formation of the ER-Sp1 complex with the wild-type but not the ER/Sp1 mutant oligonucleotide. Moreover, incubation of the wild-type bromodeoxyuridine-substituted ER/Sp1 oligonucleotide with the nuclear Ah receptor complex gave a specifically bound cross-linked 200-kDa band. These data demonstrate that Ah receptor-mediated inhibition of E2-induced cathepsin D gene expression is due to disruption of the ER-Sp1 complex by targeted interaction with an overlapping XRE.

Alkaline Phosphatase↗

PubMed1993

Mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated decrease of the nuclear estrogen receptor in MCF-7 human breast cancer cells.

Treatment of MCF-7 cells with 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 1 nM [3H]17 beta-estradiol resulted in decreased radiolabeled nuclear estrogen receptor (ER) levels as determined by velocity sedimentation analysis. In parallel studies, nuclear extracts from TCDD-treated cells also exhibited decreased binding to a consensus 32P-genomic estrogen responsive element (ERE) as determined in a gel mobility shift assay. Time-course studies showed that the decreases in nuclear ER and ER-ERE binding in TCDD-treated cells were observed within 1 to 3 h after treatment, respectively, and persisted for up to 24 h. Cycloheximide (10 microM) did not affect the TCDD-mediated response, whereas 1 microM alpha-naphthoflavone, an aryl hydrocarbon (Ah) receptor antagonist, partially blocked downregulation of nuclear ER binding by TCDD. TCDD did not significantly affect steady state ER mRNA levels as determined by Northern analysis or the rate of ER gene transcription in a nuclear run-on assay. These results suggest that the TCDD-mediated decrease in nuclear ER levels is an Ah receptor-mediated response which occurs at the translational or post-translational level.

Analysis of Variance↗

PubMed1993

What hospitals expect today from clinical systems.

Technology will always be a factor in hospital information system buys. But customers are increasingly looking for vendors that create a sincere, responsive sales relationship. In the end, the quality of support and understanding users receive can make or break a sale.

Consumer Behavior↗

PubMed1988

Thermodynamics in the prediction of the solubilities of binary mixtures of the diastereoisomers and enantiomers of ephedrine.

Equations derived from the Clausius-Clapeyron and Prigogine-Defay equations were used to determine the free energy of mixing for diastereoisomers and enantiomers from heats of fusion and transition temperature data. These equations are applied to the thermodynamics of mixing of the six binary equimolar combinations of (1R,2S)-ephedrine, (1S,2R)-ephedrine; and (1R,2R)- and (1S,2S)-pseudoephedrine isomers. The relative equilibrium solubility of the binary mixtures in water is consistent with thermodynamic calculations.

Chemical Phenomena↗

PubMed1987

Postpartum haemorrhage--a continuing problem.

The factors responsible for postpartum haemorrhage (PPH) in singleton vaginal deliveries, not complicated by a retained placenta, were identified by comparing labour characteristics in 86 women who had a PPH (blood loss greater than 500 ml) with 351 women whose blood loss at delivery was less than 350 ml. Primiparity, induction of labour by amniotomy/oxytocin, forceps delivery, long first and second stages, oxytocin compared with syntometrine (oxytocin plus ergometrine maleate), as a prophylactic oxytocic, were identified as significant risk factors. Epidural analgesia contributed indirectly to an increase in the risk of postpartum haemorrhage. The changes in labour ward practice over the last 20 years have resulted in the re-emergence of PPH as a significant problem.

Amnion↗

PubMed1986

Surfboard-related ocular injuries.

Three cases of ocular trauma caused by surfboards are reported. The severe nature of the injuries is discussed and the prevalence of this type of injury is reviewed.

Adult↗

PubMed1986

Comparison of psychomotor skills and amnesia after induction of anesthesia with midazolam or thiopental.

In two groups of 31 healthy patients undergoing minor elective surgery, anesthesia was induced intravenously with either midazolam maleate, 0.2 mg/kg, or thiopental, 3.5 mg/kg. All subjects received 2 micrograms/kg fentanyl 5 min before the induction agents. Induction time with midazolam was significantly longer than with thiopental (97.1 +/- 10.9 sec vs 59.4 +/- 5.0 sec) and time to orientation postoperatively was significantly longer after midazolam (31.7 +/- 4.2 min vs 11.0 +/- 1.1 min). Continued recovery after orientation, measured by a series of psychomotor tests, was also significantly longer with midazolam than with thiopental. Anterograde amnesia was evident in 84.8% of the midazolam treated patients and in only 31.4% of the thiopental group. This degree of absence of recall was acknowledged positively by the affected patients. The protracted recovery period may limit the use of midazolam in short surgical procedures.

Adult↗

PubMed1985

Hemodialysis clearance of pentobarbital during continuous infusion.

The hemodialysis clearance of pentobarbital during continuous infusion was determined in a 34-year-old man in acute renal failure. Pentobarbital specimens were obtained simultaneously from arterial blood entering and leaving the dialysis machine at five 1-h intervals. The mean hemodialysis clearance of pentobarbital was 22.3 ml/min. Pentobarbital concentration was relatively unaffected throughout the dialysis period, because of the high dose and continuous infusion of a drug with low hepatic intrinsic clearance and a short dialysis period. A large contribution to total body clearance was not evident, and dosage adjustment would have been unwarranted.

Acute Kidney Injury↗

PubMed1981

Hazardous materials management and control program at Oak Ridge National Laboratory--health protection.

Maintaining reasonable control of all hazardous materials used in a large research laboratory can be a formidable task. At Oak Ridge National Laboratory, a Hazardous Material Coordinator for Health Protection (HMC-Hlth) and a Hazardous Material Coordinator for Environmental Protection (HMC-Env) control hazardous materials from acquisition to disposal. The HMC-Hlth, a member of the Industrial Hygiene Department, is responsible for control of the purchase and use of hazardous materials. If the material has not had a hazard evaluation, the user is required to contact the HMC-Hlth to find out if the material is hazardous before ordering it. If the material is hazardous, the user must get permission from his divisional representative to purchase it. The user is required to fill out Part 2 of a Hazardous Material Control Card (HMCC), describing the proposed use and location of the material and to return HMCC to the HMC-Hlth. This allows the Industrial Hygiene Department to evaluate the use of the materials and to take air samples as needed. Part 1 of the HMCC also contains computer printed information on the hazards.

Chemical Industry↗

PubMed1980

Comparison of dye dilution method to radionuclide techniques for cardiac output determination in dogs.

A study was undertaken to identify the most accurate 99mTc-labeled radiopharmaceutical and to determine the accuracy of a noninvasive radionuclide technique (antecubital injection and precordial detection) for cardiac output determinations. Phase I employed sodium pertechnetate, stannous pyrophosphate with sodium pertechnetate, technetium-99m red blood cells, and technetium-99m human serum albumin as radionuclide tracers. Cardiac output was determined by the dye dilution method and then by the invasive radionuclide technique. The radiopharmaceutical was injected into the same intracardiac catheter used in the dye dilution method. Seven to 10 mongrel dogs were used to test the accuracy of each radiopharmaceutical. A paired t test and regression analysis indicated that technetium-99m human serum albumin was the most accurate radiopharmaceutical for cardiac output determinations, and the results compared favorably to those obtained by the dye dilution method. In Phase II, technetium-99m human serum albumin was used as the radionuclide tracer for cardiac output determinations with the noninvasive technique. The results compared favorably to those obtained by the dye dilution method. Regression analysis indicated a correlation coefficient of 0.91. A paired t test demonstrated that the difference between the two methods was not statistically significant (p greater than 0.05). The data suggest that a noninvasive radionuclide technique using cardiac output determinations in humans.

Animals↗

PubMed1980

The toxic materials control program at ORNL revisited.

For large industrial and research operations, maintaining reasonable control of all toxic materials used in their operations can be a formidable task. A system utilizing cards has been developed which serves a dual purpose, informing the user regarding hazards of a particular material and also facilitating appropriate workplace surveillance during its use. Selected hazard data and spill notification message, if applicable, are printed on the card. The card contains a label which the user detaches and affixes to the container. This label classifies the material according to toxicity, flammability, reactivity, special properties on a 0 through 4 hazard rating, and spill message if needed to comply with environmental requirements.

Accident Prevention↗