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R A Currie

Publications and source records attributed to R A Currie.

At least 37 records · Page 2Linked to original sources
PubMed1992

Characterization of a high affinity octamer transcription factor binding site in the human lipoprotein lipase promoter.

A high affinity octamer transcription factor (OTF-1) binding site has been identified and characterized at position--46 base pairs (bp) in the proximal human lipoprotein lipase (LPL) promoter. The affinity of the LPL OTF-1 binding site was approximately 15-fold greater than a consensus octamer sequence, ATTTGCAT, present at position--66 bp in the mouse Vk T1 promoter, and approximately 5-fold greater than the OTF-1 site present at position--49 bp in the human histone H2B promoter. Diethylpyrocarbonate interference assays have identified both 5' and 3' adenine nucleotides, which flank the core LPL ATTTGCAT sequence and interfere with OTF-1 binding when chemically modified. Introduction of mutations in either 5' or 3' flanking AT-rich sequences lowered the affinity of OTF-1 binding below the level observed with the wild-type LPL octamer oligomer. A double mutation in both flanking AT regions, however, greatly reduced the affinity of this site to levels similar to that observed with the mouse Vk T1 OTF site. An additional nuclear transcription factor, NF-Y, has been shown to bind to a functional CCAAT box motif located at -65 bp in the LPL promoter using specific alpha-NF-Y antisera. The observation of high affinity OTF-1 and NF-Y binding sites in a region of the proximal LPL promoter which is necessary for high levels of LPL transcription suggests that these sites with their associated proteins play important functional roles in the transcriptional activation of the LPL promoter during adipocyte differentiation.

3T3 Cells↗
PubMed1992

Determination of transcription factor isoelectric point by two-dimensional native isoelectric focusing and electrophoretic mobility shift analysis.

A rapid, sensitive, and inexpensive assay is described for the determination of isoelectric points of native transcription factors derived from cell nuclei. This assay depends on a transcription factors' ability to bind DNA with high specificity and obviates the need for specific antisera or additional detection methods in identifying a particular protein following isoelectric focusing. This method has been applied to two ubiquitous proteins, the octamer transcription factor, Oct-1, and the multisubunit CCAAT box factor, NF-Y. Isoelectric points have been determined under native conditions using conventional isoelectric focusing in the first dimension, followed by binding to a specific oligonucleotide DNA probe, and separation of specific DNA/protein complexes from unbound DNA in the second dimension using native polyacrylamide gel electrophoresis. Oct-1 from HeLa cell nuclei was shown to have a pI of 9.6, while NF-Y was shown to have a pI of 4.5. This method is applicable to any transcription factor which binds DNA specifically and may be used to identify changes in surface charge characteristics which occur as a result of alternative splicing events and/or following transcription factor post-translational modification(s).

Base Sequence↗
PubMed1990

Regulation of kappa immunoglobulin gene transcription in vitro.

An in vitro transcription system has been established with a whole-cell extract from the human Burkitt's lymphoma, Daudi, cell line. The B cell extract has been compared with a HeLa cell extract in an effort to study lymphocyte-specific regulatory factors of kappa light chain gene transcription. Both extracts were capable of transcribing Vk genes and other RNA polymerase II dependent genes. Alpha-amanitin at [0.1 micrograms/ml] completely inhibited the accumulation of transcripts in both systems. At low DNA template concentrations the kappa intronic enhancer stimulated Vk promoter transcription 3-7 fold in B cell extracts. The enhancer-dependent transcription was abolished by excising the enhancer from the test plasmid with Eco R1. Both Vk promoter and enhancer-dependent transcription in HeLa extracts was undetectable at low [DNA]. These results demonstrate that kappa enhancer stimulation of Vk transcription in vitro is observed in B cell extracts only under conditions of low DNA template concentration.

Amanitins↗
PubMed1989

Identification of an octamer-binding site in the mouse kappa light-chain immunoglobulin enhancer.

A 215-base-pair (bp) region of the mouse MOPC 41 kappa light-chain immunoglobulin gene enhancer has been analyzed for specific binding of lymphoid and nonlymphoid nuclear factors. Mobility shift assays with a series of overlapping DNA fragments have mapped DNA-binding sites for three unique factors. The B-cell-specific (OTF-2) and ubiquitous (OTF-1) octamer-binding transcription factors specifically bound to a site centered about 136 bp 5' of the nuclear factor NF-kappa B site. A third specific factor, NF-kappa E, bound to a site that was about 75 bp 5' of the NF-kappa B site and within a region important for enhancer function. This novel factor was found in both mature B and HeLa cell nuclei. B-cell OTF-2, B-cell OTF-1, and HeLa OTF-1 bound to the kappa enhancer and kappa promoter octamer sites with similar affinities despite a 2-bp difference in the kappa enhancer octamer sequence. However, DNase I footprint analyses indicated that affinity-purified OTF-2 bound both to the enhancer OTF site and, surprisingly, to 80 bp of A + T-rich flanking sequence. Moreover, methylation interference studies demonstrated distinct differences in OTF interactions between the consensus octamer in the kappa promoter and the nonconsensus octamer identified in the enhancer. This novel observation of an OTF-binding site in the kappa enhancer provides a common link with the OTF sites in the promoter-proximal regions of all kappa promoters and thus mirrors the structural arrangement of OTF sites found in the promoters and enhancers of immunoglobulin heavy-chain genes.

Animals↗
PubMed1986

Regulation of epidermal growth factor receptors by glucocorticoids during the cell cycle in HeLa S3 cells.

Glucocorticoids have been shown to increase epidermal growth factor (EGF) receptors in HeLa S3 cells via mechanisms dependent upon glucocorticoid receptors. We have now examined the basal and glucocorticoid-induced levels of epidermal growth factor (EGF) receptors in synchronized HeLa S3 cells and related these findings to glucocorticoid receptor nuclear binding, receptor activation, and several physiochemical properties of the glucocorticoid receptor. Quantitation of EGF receptor binding during the cell cycle indicates that no significant variation in EGF receptor number occurs during the cell cycle. Dexamethasone treatment of nonsynchronized HeLa S3 cells results in an approximately 131% increase in EGF receptor number within 48 h of treatment. Administration of glucocorticoids to cells synchronized at the late G1/S phase boundary of the cell cycle results in an approximately 80% increase in epidermal growth factor receptors 8-9 h after treatment. This hormone-induced response disappears as cells enter the G2/M and early G1 phases of the cell cycle. In contrast, hormone administration to synchronized cells during the G2/M phases is without effect after 8 or 9 h, but a response is evident when these cells reenter the late G1 phase. This inability of glucocorticoids to induce EGF receptor binding has been correlated with nuclear glucocorticoid receptor translocation at 37 degrees C in intact cells and activation of receptors in vitro to DNA binding proteins by warming. This reduction in nuclear receptor binding in intact cells and diminished in vitro activation of receptor are associated with the detection of a tightly binding glucocorticoid receptor form as analyzed by hydroxylapatite chromatography. These analyses suggest that the failure of glucocorticoids to induce EGF receptor binding during the G2/M and early G1 phases may be the result of decreased receptor activation which may result from a post-transcriptional modification of the glucocorticoid receptor.

Cell Cycle↗
PubMed1984

The physicochemical nature of 37 degrees C cytoplasmic glucocorticoid receptors in HeLa S3 cells.

The physicochemical properties of size, shape and surface charge have been determined for the soluble fraction of cytoplasmic glucocorticoid receptors which are located in the HeLa S3 cell cytoplasm after incubation of whole cells with glucocorticoid at 37 degrees C. Under hypotonic buffer conditions approximately 80% of the total recovered [3H]triamcinolone acetonide receptor complexes sedimented through a 5-20% density gradients to the tube bottom, and approximately 90% eluted from a Sephacryl S-300 gel exclusion column in the void volume. Increasing the [KCl] of the buffer in the sucrose density gradients, and gel exclusion columns to 0.15 M caused a reduction in the percentage of this large aggregate to approximately 64% and approximately 75%, respectively. Further increases in the [KCl] during analysis to 0.4 M reduced the percentage of rapidly sedimenting receptors to approximately 62%, and shifted the sedimentation coefficient of the slower sedimenting receptors from approximately 5.2 S to 3.9 S. These conditions also decreased the fraction of receptor in the void volume of gel exclusion columns to 67%. Ion exchange analysis of receptor binding to DEAE cellulose, hydroxylapatite, phosphocellulose, and DNA cellulose revealed heterogenous populations of receptor species; comprising both "unactivated" and "activated" receptor forms. The ratios of unactivated/activated receptors was highly dependent on the matrix employed and differed substantially among those evaluated. For example, by the criteria of DEAE cellulose and phosphocellulose chromatography approximately 60% of the total 37 degrees C cytoplasmic receptors were in the "activated" state. A large fraction of these receptors, however, failed to bind to DNA cellulose. These results demonstrate that the glucocorticoid receptors which remain in the HeLa S3 cytoplasm at 37 degrees C do not bind to ion exchange materials, which are used as indexes of receptor "activation," in a uniform manner. We hypothesize that the diminished DNA binding capability of these receptors accounts for their cellular localization in the HeLa S3 cell cytoplasm at 37 degrees C.

Centrifugation, Density Gradient↗
PubMed1983

Comparison of pyridoxal phosphate and 0.4 M KCl-extracted nuclear glucocorticoid receptors in HeLa S3 cells.

A comparison of the physicochemical properties between pyridoxal 5'-phosphate- and 0.4 M KCl-extracted nuclear glucocorticoid receptors has been made utilizing HeLa S3 cells as a source of receptor. Both pyridoxal 5'-phosphate/NaBH4-reduced and 0.4 M KCl-extracted receptors sedimented as approximately 3.5-4.5 S species in 5-20% sucrose gradients containing 0, 0.15, and 0.4 M KCl. Under low-ionic-strength buffer conditions, pyridoxal 5'-phosphate-extracted receptor elutes close to the void volume of a Sephacryl S-300 gel-exclusion column. Increasing the [KCl] of the column to 0.4 M resulted in the elution of receptor with a Stokes radius of 58 A and calculated Mr = 96,000. Nuclear receptors extracted with 0.4 M KCl also formed a large-molecular-weight complex which eluted close to the void volume of the gel-exclusion column. Increasing the [KCl] to 0.4 M had the effect of shifting this receptor form to a species which had a Stokes radius of 62 A and calculated Mr = 89,700. Ion-exchange analysis of nuclear-extracted receptors revealed that 0.4 M KCl-extracted receptors exhibited considerable charge heterogeneity, whereas pyridoxal 5'-phosphate-extracted receptors did not. Pyridoxal 5'-phosphate-extracted receptors (approximately 86%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M; approximately 14% of the receptors had little affinity for DEAE-cellulose. Pyridoxal phosphate-treated receptors had little affinity for hydroxylapatite, phosphocellulose, and DNA-cellulose. The predominant form of 0.4 M KCl-extracted nuclear receptors (approximately 78%) eluted from DEAE-cellulose between 0.05 and 0.15 M KCl, a position coincident with "activated" glucocorticoid receptors. The remaining receptor fraction (approximately 22%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M, a position coincident with "unactivated" glucocorticoid receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Nucleus↗
PubMed1983

Determination of 2-chloroethanol in honey, beeswax, and pollen.

Procedures were developed and tested for the determination of residues of 2-chloroethanol (ethylene chlorohydrin, ECH) in honey, beeswax, and pollen. Recoveries of ECH from fortified samples averaged 91, 87, and 89%, respectively, for each substrate. The maximum amount of ECH found in substrate fumigated with ethylene oxide was 36 micrograms/g in honey, 124 micrograms/g in beeswax, and 132 micrograms/g in pollen. A tendency was noted for darker waxes, which contain larger amounts of naturally occurring chlorides than light-colored waxes, to contain the greater amounts of ECH. A gas-liquid chromatograph equipped with a Dohrmann halogen-specific detector was used for identification and quantitation.

Animals↗
PubMed1982

Physicochemical properties of the cytoplasmic glucocorticoid receptor complex in HeLa S3 cells.

The physiochemical properties, size, shape, and surface charge, have been determined for the cytoplasmic glucocorticoid receptor complex (GR) in randomly growing HeLa S3 cells. Sucrose density gradient and gel exclusion chromatographic analysis have shown GR to undergo a marked reduction in size and shape when analyzed under increasing KCl concentration conditions. Analysis of GR prepared in a hypotonic buffer solution revealed a large, 7-8 S species with Ve/Vo ratio of 1.15, and a Stokes radius of congruent to 95 A. Increasing the KCl concentration in the analysis buffer to 0.15 resulted in reduction in GR size and shape to a 4.5 S species with the following properties: Ve/Vo = 1.38. Stokes radius = 69 A. and calculated Svedberg molecular weight = 132,000. A limit to the observed decrease in GR size and shape was obtained under hypertonic, 0.4 M KCl, conditions; a 3.75 S form was observed to elute from the gel exclusion column with a Ve/Vo = 1.40. Stokes radius = 65 A. and a calculated Svedburg molecular weight = 102,000. Exposure to higher KCl conditions, 0.6 M and 0.8 M, resulted in no further decrease in GR size or shape. Ion exchange chromatographic analysis of cytoplasmic GR revealed heterogeneous populations of GR with apparent differences in surface charge. GR binding to DEAE cellulose revealed a predominant form which eluted at 0.15 M KCl (Form I). Under both hypotonic and hypertonic conditions small populations of GR forms were observed to elute from DEAE at or approximately 0.1 M KCl (Form III), and or approximately 0.3 M KCl (Form II), respectively. Binding of GR to hydroxylapatite (HAP) confirmed the heterogenous nature of GR. Cytoplasmic GR partitioned into at least 3 forms om HAP. The predominant GR form eluted from HAP at 0.1 M K2HPO4 (Form I); secondary, and tertiary forms eluted at 0.125 M (Form II), and 0.15 M K2POH4 (Form III), respectively. Glucocorticoid receptors were observed to elute from columns of phosphocellulose, and DNA cellulose in the void volume. Two receptor forms were observed with isoelectric focusing of HeLa S3 cytoplasm in Sephadex IEF G-75 gels. The primary, and secondary species had pI values of 7.5 and 6.3, respectively. These results demonstrate that randomly growing HeLa S3 cells contain heterogeneous population of GR in the cytoplasmic compartment. Randomly growing HeLa S3 cell cytoplasm appears to contain two "unactivated" receptor species (Forms I and II) that differ in their overall surface charge properties; a third activated species (Form III) appears to arise from heat induced "activation" during cell homogenization, and is more basic in nature.

Cell Cycle↗
PubMed1982

Pyridoxal phosphate blocks aggregation of molybdate treated glucocorticoid receptors in HeLa S3 cells.

We have compared the effects of sodium molybdate and pyridoxal phosphate on the sedimentation properties of cytoplasmic glucocorticoid receptors. Whole HeLa S3 cells were incubated with [3H]-Dexamethasone (Dex) at 0-4 degrees C to produce unactivated cytoplasmic steroid receptors. When these cells are lysed in 10 mM Tris, 1 mM EDTA, 12 mM alpha-thioglycerol pH 7.4 (buffer A), [3H]-Dex receptor sediment as 7-8S species in sucrose gradients prepared in the same buffer. Sedimentation of receptors on gradients containing buffer A plus 20 mM sodium molybdate (buffer B) results in an approximately 9S species. Treatment of these receptors with pyridoxal phosphate (10 mM) followed by reduction with NaBH4 and sedimentation on sucrose gradients prepared in either buffer A or B results in the production of 3-4S species. Although [3H]-Dex labeled cells lysed in buffer B yield approximately 9S species when analyzed in gradients prepared in buffer B, centrifugation of these receptors in buffer A yield 7-8S species. Similarly when cells are lysed in buffer A labeled receptors sediment in sucrose gradients prepared in buffer B as approximately 9S species. Receptors prepared in buffer B and treated with pyridoxal phosphate and NaBH4 sediment in buffer B gradients as two discrete approximately 7S and approximately 3.5S species. Pyridoxal phosphate and NaBH4 treated receptors prepared in buffer B sediment as disperse 3-6S species when analyzed in buffer A gradients. Based on these observations we conclude that pyridoxal phosphate, NaBH4 reduced receptors are not particularly susceptible to subsequent molybdate action, but molybdate pre-treated receptors can be influenced by pyridoxal phosphate.

Centrifugation, Density Gradient↗
PubMed1982

Identification of modified forms of human glucocorticoid receptors during the cell cycle.

Glucocorticoids induce alkaline phosphatase activity in HeLa S3 cells only during the Late G1 and S phases of the cell cycle. In order to determine why these cells respond to glucocorticoids only during these phases, we have determined the physicochemical characteristics of the glucocorticoid receptor (GR)1 in synchronized HeLa S3 cells. GR isolated from HeLa S3 cells in the Late G1 and S phases elute from DEAE cellulose as a single, major peak at a competing [KCl] of 0.15 M (Form I). In contrast, GR isolated from cells in the G2 and Early G1 phases show a biphasic elution pattern; a more highly acidic GR form elutes at a [KCl] or 0.25 M (Form II). Similar elution patterns are observed when GR is chromatographed on hydroxylapatite (HAP). In these studies a [K2HPO4] of 0.125 M elutes the major GR form in Late G1 and S phase cells, whereas a [K2HPO4] of 0.15 M elutes a second binding species from G2 and Early G1 phase cells. GR sediments through 5-20% sucrose density gradients as an approximately 4.6S species under 0.15 M KCl buffer conditions in all phases of the cell cycle; thus the altered receptor charge is not accompanied by a change in receptor size or shape. These data suggest that HeLa S3 cell GR are susceptible to modification during the cell cycle. Conversion of GR to acidic forms may relate to the receptors' ability to transmit hormonal signals.

Cell Cycle↗
PubMed1980

Extraction of pentachlorophenol and tetrachlorophenol residues from field-contaminated carrots and potatoes: comparison of several methods.

A number of solvent extraction systems were investigated for their efficiency in removing residues of pentachlorophenol (PCP) and tetrachlorophenol (TCP) from field-contaminated carrots and potatoes. Greatest numerical yields of PCP and TCP, for both carrots (9 and 5 ng/g, respectively) and potatoes (147 and 11 ng/g, respectively), were obtained by Soxhlet extraction with acidified acetone for 44 hr. Within the limits of experimental error, equivalent results could be obtained by using a 20 hr Soxhlet extraction with acidified acetone for carrots, and a 5 min blending with acidified acetone for potatoes. Extracts were reacted with diazoethane to form the ethyl ethers of PCP and TCP, followed by cleanup on a Florisil column. A gas-liquid chromatograph equipped with a 63Ni linearized electron capture detector was used for identification and quantitation.

Chlorophenols↗
PubMed1979

Pesticide residues in human milk, Alberta, Canada--1966-70, 1977-78.

Organochlorine pesticide residues were determined in human milk of residents of Alberta, Canada, during 1966-70 and 1977-78. Levels of polybrominated biphenyls (PBBs), polychlorinated biphenyls (PCBs), and some common organophosphorus pesticides were also monitored during 1977-78. Average residue levels were generally lower in 1977-78 samples, whereas the percent incidence of residues was generally lower in 1966-70 samples. beta-BHC was found in all 1977-78 samples, but was not detected in 1966-70 samples. PCBs were detected in all but two of the 1977-78 samples. Average levels of p,p'-DDT and is metabolites were substantially lower during the second period than during the first. Large increases in the average levels and percent incidences of heptachlor epoxide and o,p'-DDT have been attributed to refinements in both cleanup and gas chromatographic analysis. Although no PBBs or organophosphorus pesticides were detected in 1977-78 samples, an unidentified hydrocarbon similar to dicofol was found in all samples of that period. No correlation between donor age groups and average pesticide residue levels could be inferred.

Adult↗