Biomedical subjects

J H Clark

Publications and source records attributed to J H Clark.

At least 91 records · Page 5Linked to original sources

PubMed1991

Effects of feeding alkaline hydrogen peroxide-treated wheat straw-based diets on intake, digestion, ruminal fermentation, and production responses by mid-lactation dairy cows.

The objective of this experiment was to determine the effects of feeding different levels of alkaline hydrogen peroxide-treated wheat straw (AHP-WS) in the diet on feed intake, nutrient digestion, ruminal fermentation, and production responses in mid-lactation dairy cows. Eight Holstein cows, averaging 147 d postpartum, were used in two replications of a 4 x 4 Latin square design. Complete mixed diets consisted of 70% forage and 30% concentrate (DM basis) with various levels of AHP-WS, alfalfa haylage, and corn silage as forage sources. Treatments contained 0 (control), 20.0, 40.1, or 60.0% AHP-WS in the diet. A quadratic effect (P = .08) of AHP-WS level on DMI was noted, with values of 2.16, 22.3, 20.8, and 18.9 kg/d for the control, 20.0, 40.1, and 60.0% AHP-WS treatments, respectively. Apparent digestibilities of DM, OM, CP, and ADF were not affected (P greater than .10) by replacing haylage and corn silage with increasing amounts of AHP-WS in the diet, but there was a linear increase (P = .03) in NDF digestibility (44% for control vs 59% for the 60.0% AHP-WS diet) and a parallel decrease (P less than .05) in cell content digestibility (82 vs 70% for these two diets). Yields of milk and 4% fat-corrected milk (FCM) were decreased (quadratic; P = .0001) as the level of AHP-WS increased in the diet. The addition of AHP-WS to the diet decreased the milk fat percentage from 3.72 to 3.60% (quadratic; P = .05) and decreased milk protein percentage from 3.27 to 3.13% (linear; P = .0001). Cows fed the higher levels of AHP-WS had linear increases (P = .0001) in ruminal concentrations of total VFA (128.0 mM for control vs 136.0 mM for the 60.0% AHP-WS treatment) and molar proportion of acetate, resulting in a quadratic effect (P less than .0001) on the acetate:propionate ratio. These data indicate that feeding the 40.1 and 60.0% AHP-WS diets lowered digestible DM and OM intakes, which resulted in reduced 4% FCM yield as nutrient intakes were decreased compared with cows fed the 20.0% AHP-WS diet or the control diet containing alfalfa haylage and corn silage. Although substituting AHP-WS for haylage and corn silage increased NDF digestibility and tended to increase digestible NDF intake, milk production was depressed because digestible DMI decreased.

Animal Feed↗

PubMed1991

Anticholinesterase activity of potential therapeutic 5-(1,3,3-trimethylindolinyl) carbamates.

Six N-alkyl and N-aryl 5-(1,3,3-trimethylindolinyl) carbamates were synthesized and studied for their structure-activity relationships in inhibiting eel acetylcholinesterase (AChE). The carbamates were 5-(1,3,3-trimethylindolinyl)N,N-dimethylcarbamate (Cui Xing Ning) (I), 5-(1,3,3-trimethylindolinyl)N,N-diethylcarbamate (IV), 5-(1,3,3-trimethylindolinyl)N-ethylcarbamate (III), 5-(1,3,3-trimethylindolinyl)N,N-diethylcarbamate (IV), 5-(1,3,3-trimethylindolinyl)N-heptylcarbamate (V), and 5-(1,3,3-trimethylindolinyl)N-(3-chlorophenyl)carbamate (VI). The inhibition studies were carried out at 25.0 degrees C at pH 7.60. The rank order of the ki values for eel AChE inhibition is II > V > I > III > VI > IV. Compound II has a greater affinity for the enzyme than any irreversible inhibitor cited in the literature (Kd = 7.14 x 10(-8) M). Our findings should aid in the application of these carbamates (1) for counteracting the cholinergic problems associated with various diseases, and (2) for developing potential pretreatment compounds for organophosphate poisoning.

Acetylcholinesterase↗

PubMed1991

Effects of urea and starch on rumen fermentation, nutrient passage to the duodenum, and performance of cows.

Four midlactation, multiparous Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square design to determine the effects of supplementing urea or starch or both to diets containing fish meal on passage of nutrients to the small intestine and performance of lactating cows. The treatments (in a 2 x 2 factorial arrangement) were 1) control and control plus 2) urea, 3) starch, or 4) starch and urea. Supplementing diets with urea did not affect DMI; ruminal, postruminal, or total tract digestibilities of DM, starch, ADF, or NDF; ruminal fluid VFA concentrations or molar percentages; or ruminal fluid or particulate dilution rates. Feeding additional starch depressed DMI but did not alter ruminal or postruminal digestion of OM or VFA concentrations and molar percentages in ruminal fluid. Ruminal fluid ammonia concentration was increased by feeding urea and decreased by feeding additional starch. Passage of nonammonia N, nonammonia nonmicrobial N, or microbial N to the small intestine and efficiency of microbial CP synthesis were not affected significantly by supplying either urea or additional starch. Feeding urea increased passage of methionine to the small intestine, whereas feeding additional starch increased passage of methionine and arginine. Passage of other amino acids to the small intestine was not altered significantly by feeding urea or additional starch. Production of milk and milk protein was increased, but yields of fat and SNF were not altered by feeding diets supplemented with urea. Production of milk and milk fat was not affected, but yields of CP and SNF were decreased when additional starch was fed to cows.

Ammonia↗

PubMed1991

Effects of calcium salts of fatty acids and protein source on ruminal fermentation and nutrient flow to duodenum of cows.

Four Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square to investigate the effects of calcium salts of long-chain fatty acids (fat) and source of protein (fish meal or soybean meal) on ruminal fermentation, flow of nutrients to the small intestine, and animal performance. Cows were fed for ad libitum intake a diet of 30% alfalfa haylage, 20% corn silage, and 50% concentrate on a DM basis. Treatments, arranged in a 2 x 2 (fat x protein) factorial, were 1) soybean meal, no fat; 2) soybean meal, fat; 3) fish meal, no fat; and 4) fish meal, fat. Intake of DM was not affected by fat or protein source, but feeding fat decreased the amount of OM truly digested in the rumen. Starch intake was decreased, but flow of starch to the duodenum was not altered by feeding fat. Nonammonia N and microbial N flows to the duodenum were not affected by treatment comparisons. However, efficiency of microbial growth was increased by feeding fat, but not by source of protein. Passage of amino acids to the duodenum was not affected by source of protein, probably because fish meal contributed only 17% of the total dietary CP, and microbial N constituted about 50% of the NAN passing to the duodenum; this had an equalizing effect on the pattern and quantity of amino acids that passed to the duodenum. Feeding fat or different sources of protein did not alter milk production. Milk fat percentage was increased, and protein percentage was decreased when fat was fed, but yields of milk fat and protein were not different.

Amino Acids↗

PubMed1991

Effects of calcium salts of fatty acids and proportion of forage in diet on ruminal fermentation and nutrient flow to duodenum of cows.

Four Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square to investigate the effects of calcium salts of long-chain fatty acids (fat) and proportion of forage in diet on ruminal fermentation, flow of nutrients to the small intestine, and animal performance. Treatments, arranged in a 2 x 2 (fat x forage) factorial, were 1) low (50%) forage, no fat; 2) low forage, fat; 3) high (67%) forage, no fat; and 4) high forage, fat. Feeding fat decreased OM intake and OM truly digested in the rumen. Feeding high forage diets decreased intakes of OM and starch and increased intakes of ADF and NDF. Ruminal pH and ratio of acetate to propionate were increased with high forage diets compared with low forage diets. Feeding fat and different amounts of forage to cows did not alter the flows of NAN and microbial N to the duodenum or efficiency of microbial growth. Production of milk and 4% FCM and percentage of fat in milk were increased by feeding fat. Feeding high forage diets decreased milk production, increased percentage of fat in milk, increased the yield of fat, and caused no change in 4% FCM production. The percentage of protein in milk was decreased by feeding high forage diets and fat, but yield of milk protein was decreased only by feeding high forage diets to cows.

Amino Acids↗

PubMed1991

Responses of dairy cows to supplemental rumen-protected forms of methionine and lysine.

Multiparous Holstein cows at six universities were utilized to examine effects of ruminally protected methionine and lysine on lactational performance. Three hundred and four cows began the study; 259 cows were included in the production analysis. Following a 21-d standardization period, cows received a basal diet of corn silage and ground corn supplemented with one of five dietary treatments, which were supplements of soybean meal or corn gluten meal, the latter with zero and three combinations of protected methionine and lysine (methionine; methionine and lysine; methionine and double (2x) lysine). Treatment effects were evaluated during early, mid, late, and total lactation (22 to 112, 113 to 224, 225 to 280, and 22 to 280 d postpartum, respectively). On a DM basis, ratios of forage to concentrate (50:50, 60:40, and 70:30) increased, and dietary CP (16.0, 14.5, and 13.0%) decreased during the three periods of lactation. Amount of amino acid supplementation also decreased (15, 12, and 9 g/d methionine; 20, 16, and 12 g/d lysine; and 40, 32, and 24 g/d 2x lysine) with period of lactation. Actual and least squares means for milk, FCM, and milk protein yields were greater for soybean than for corn gluten meal during early, mid, and total lactation. In addition, these variables responded linearly to lysine in early lactation. Response to lysine was quadratic during mid and total lactation for these variables. Differences in nutrient intake explained production responses to protein sources but not to lysine. Serum amino acid responses primarily reflected differences in dietary protein source and rumen-protected amino acid.

Amino Acids↗

PubMed1991

Effects of dietary fat and protein on fatty acid flow to the duodenum and in milk produced by dairy cows.

Four Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square with treatments in a 2 x 2 factorial arrangement. Treatments were 1) soybean meal, no fat; 2) fish meal, no fat; 3) soybean meal, fat; and 4) fish meal, fat. Cows were fed for ad libitum intake a diet of alfalfa haylage, corn silage, and concentrate (30:20:50) on a DM basis. Intake of gross energy (105 Mcal/d) was not altered by treatment comparisons. However, feeding fat decreased energy digested in the rumen (15 vs. 24%) and increased energy digested postruminally (55 vs. 43%) but resulted in similar amounts of energy (72 Mcal/d) digested in the total tract. The flow of C14:0, C16:0, C18:0, C18:1, C18:2, C18:3, and total fatty acids to the duodenum was increased by feeding fat. The average flow of C14:0, total C18, and total fatty acids to the duodenum was greater than their intake for all treatments, suggesting de novo synthesis of fatty acids by ruminal microbes. Biohydrogenation of unsaturated C18 was decreased 70, 67, 59, and 51% for treatments 1 to 4, respectively, by feeding fat and fish meal. Digestibility of total fatty acids entering the small intestine (78%) was not altered by treatment comparisons; however, feeding fat altered digestibility of individual fatty acids. The proportion of C16:0 and C18:1 was increased, and the proportion of C6:0, C8:0, C10:0, C12:0, and C14:0 was decreased in milk fat produced by cows fed fat.

Animal Feed↗

PubMed1991

Effects of somatotropin and duodenal infusion of amino acids on nutrient passage to duodenum and performance of dairy cows.

Four multiparous Holstein cows were used in a 4 x 4 Latin square to investigate the effects of bST and postruminal infusion of lysine and methionine on ruminal fermentation, flow of nutrients to the small intestine, and animal performance. The treatments were 1) control; 2) control plus 24 g of lysine and 8 g of methionine/d; 3) control plus 25 mg of bST/d; and 4) control plus 25 mg of bST/d plus 24 g of lysine and 8 g of methionine/d. Intakes of DM, OM, CP, starch, NDF, and ADF were similar among treatments. Ruminal characteristics, flow of nutrients to the small intestine, and total tract apparent digestibilities of nutrients were not affected by injection of bST or postruminal infusion of lysine and methionine in this short-term experiment. Milk production, 4% FCM, milk fat percentage and yield, and production of milk CP were increased by administering bST. Postruminal infusion of lysine and methionine did not affect milk production or composition.

Ammonia↗

PubMed1990

Methyl p-hydroxyphenyllactate and nuclear type II binding sites in malignant cells: metabolic fate and mammary tumor growth.

Previous studies in our laboratory demonstrated that methyl p-hydroxyphenyllactate (MeHPLA) is an important cell growth-regulating agent which binds to nuclear type II binding sites in normal and malignant cells. Furthermore, this compound is deficient in a variety of rat and mouse mammary tumors and human breast cancer preparations, and this deficiency correlates with the loss of regulatory control. The present studies were performed to examine the metabolic fate of [3H]MeHPLA in mouse mammary tumors. Stable analogs of this compound such as 4,4'-dihydroxy benzylidene acetophenone were also assessed for nuclear type II site binding affinity and their ability to inhibit mammary cancer cell growth and proliferation in vitro and in vivo. The results demonstrate that mouse mammary tumors contain esterase activity which hydrolyzes MeHPLA to p-hydroxyphenyllactic acid, and this was the only major metabolite detected in these tumor preparations in vitro or in vivo. 4,4'-Dihydroxy benzylidene acetophenone, an esterase-stable MeHPLA analog, was found to bind with high affinity to nuclear type II sites but not the estrogen receptor, was capable of occupying type II sites in cultured MCF-7 cells, and inhibited the proliferation of these cells in concentrations which directly correlated with type II binding site occupancy. Similarly, 4,4'-dihydroxy benzylidene acetophenone administration by silastic implant or injection resulted in a dose-dependent inhibition of the growth of transplantable mammary tumors in mice, suggesting that this stable analog mimicks MeHPLA as a cell growth-regulating agent. Taken together, these results suggest esterase hydrolysis of MeHPLA in mammary tumors may result in a deficiency in this compound which correlates with a loss of regulatory control.

Animals↗

PubMed1990

High level expression of a truncated chicken progesterone receptor in Escherichia coli.

Using a novel Escherichia coli system we have successfully overexpressed a region of the chicken progesterone receptor which encodes both the DNA- and hormone-binding domains. The expression system produces the truncated receptor fragment as an in-frame fusion with ubiquitin. This strategy greatly enhances both the solubility and stability of fusion proteins expressed in E. coli. Synthesis has been further improved by induction of the lambda PL promoter with nalidixic acid at low growth temperatures (less than or equal to 30 degrees C) rather than use of conventional heat induction protocols. We can produce 10 mg of receptor fragment/liter of cells using this system, and we estimate that at least 0.3 mg of this receptor material is biologically active, as assessed by DNA-binding and hormone-binding assays. Receptor produced in this manner is almost indistinguishable from authentic oviduct progesterone receptor using the criteria of hormone-binding specificity and affinity and binding to a progesterone response element. This expression system offers a cheap convenient method for the production of mg amounts of biologically active derivatives of progesterone receptor for biochemical studies.

Animals↗

PubMed1990

Formation of methemoglobin and metmyoglobin using 8-aminoquinoline derivatives or sodium nitrite and subsequent reaction with cyanide.

The kinetics of the oxidation of hemoglobin (Hb) and myoglobin (Mb) by sodium nitrite, 8-[(4-amino-1-methylbutyl)amino]-6-methoxy-quinoline diphosphate (primaquine), 6-methoxy-8-(6-diethylaminohexylamino)-4-methyl-quinoline dihydrochloride (WR6026) and 8-[(4-amino-1-methylbutyl)amino]-2,6-dimethoxy-4-methyl- 5-[(3-trifluoromethyl)phenoxy]quinoline succinate (WR238,605) were studied at pH values ranging from 7.4 to 7.6 and at 37 +/- 1 degrees C. The reaction between Hb and primaquine, WR6026 and WR238,605 resulted in precipitation, as did the reaction between Mb and WR238,605. The reaction between nitrite ion (NO2-) and Hb showed a lag period followed by an autocatalytic phase. The data in this study are consistent with and substantiate the proposed mechanism for the Hb-NO2- oxidation reaction. The reaction between Mb and NO2- at higher NO2- concentrations also showed a lag period followed by an autocatalytic period, while at lower NO2- concentrations no lag period was seen. The data suggest a shift in rate constant at these lower NO2- concentrations. The reaction between Mb and both WR6026 and primaquine followed a two-term rate law with oxidant-dependent and -independent terms. Concentration-effect curve data, along with these results, suggest the presence of a catalytic pathway. The rates of formation of cyanomethemoglobin and cyanometmyoglobin complexes from cyanide ion and methemoglobin (MHb) and metmyoglobin (MMb), respectively, were followed in the presence of the heme oxidants. The rate constants were all within a narrow range and suggest that complexation of cyanide by MHb and MMb is not affected by the presence of oxidants.

Aminoquinolines↗

PubMed1990

pH effects in the spontaneous reactivation of phosphinylated acetylcholinesterase.

Previous studies on the spontaneous reactivation of phosphorylated and phosphonylated cholinesterases report bell-shaped curves with reaction rate maxima between pH values of 7 and 9. By way of contrast, we found reactivation rate minima in the same pH region for a phosphinylated bovine erythrocyte acetylcholinesterase and three phosphinylated eel acetylcholinesterases. To further elucidate these observations, eel acetylcholinesterase was inhibited with racemic 4-nitrophenyl ethyl(phenyl)phosphinate. The spontaneous reactivation of the inhibited enzyme over the pH range 6.00 to 9.00 was monitored following 1. both inhibition and spontaneous reactivation at the same pH, and 2. inhibition at pH 7.60 followed by spontaneous reactivation at the selected pH. The combined plots of both studies gave overlapping pH curves with minima around pH 7.60. The results indicate that the minima in the rates of the spontaneous reactivation of phosphinylated acetylcholinesterases are not the consequence of a pH-controlled change in the relative inhibition rates of the P(+)- and P(-)-enantiomers participating in the inhibition reaction. Our results suggest that the orientation of the phosphinyl group in the active site of phosphinylated acetylcholinesterase is quite different from that of the inhibitor groups in phosphonylated or phosphorylated enzyme.

Acetylcholinesterase↗

PubMed1990

Relationship between ovarian activity and energy status during the early postpartum period of high producing dairy cows.

The effect of energy status upon ovarian activity during early lactation was assessed in 54 multiparous Holstein cows. Dry matter intake and milk yield were measured daily from parturition through 9 wk of lactation. Milk composition and body weight were measured weekly during this time. Energy balance corrected for body weight loss was calculated weekly. Blood was collected via tail vein three times weekly and plasma analyzed for progesterone and nonesterified fatty acids. Fifteen cows were anestrus for the 9 wk based on their plasma progesterone (less than 1 ng/ml). These cows were compared with two cycling groups, one group of 25 cows showing corpus luteum activity within 40 d after parturition and a second group of 14 cows showing corpus luteum activity between 40 and 60 d postpartum. Anestrus cows ate less feed, produced less milk, and lost more body weight, resulting in a more negative energy status than cycling cows. Differences in energy balance among cow groups were greatest the first 3 wk postpartum. Anestrus cows and cows showing corpus luteum activity between d 40 and 60 obtained more energy from body reserves for milk production the first 2 wk of lactation, than cows cycling prior to d 40.

Anestrus↗

PubMed1990

Effects of source and amount of protein on ruminal fermentation and passage of nutrients to the small intestine of lactating cows.

Four Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square to investigate the effects of source (corn gluten meal or soybean meal) and amount (14.5 or 11.0%) of CP on ruminal fermentation, passage of nutrients to the small intestine, and animal performance. Cows wee fed for ad libitum intake a diet of 60% corn silage and 40% concentrate on a DM basis. The treatments, arranged in a 2 x 2 (source x amount of CP) factorial, were 1) 14.5% CP, soybean meal; 2) 11.0% CP, soybean meal; 3) 14.5% CP, corn gluten meal; and 4) 11.0% CP, corn gluten meal. Digestion in the rumen of OM, starch, ADF, and NDF was not affected by source or amount of CP in the diet. Total VFA and NH3 concentrations in ruminal fluid were increased by feeding diets that contained 14.5% CP or soybean meal. FLows of non-NH3 N and amino acids to the duodenum were greater in cows fed the 14.5% CP diets because of a greater flow of non-NH3 nonmicrobial N to the duodenum. Larger amounts of lysine passed to the duodenum when cows were fed soybean meal compared with corn gluten meal. Microbial N flow to the duodenum and efficiency of microbial growth were not affected by treatments, suggesting that ruminal NH3 concentration was not limiting for maximal microbial protein synthesis. Feeding 14.5% CP diets increased the production of milk (29.5 vs. 26.8 kg/d) and milk protein compared with 11.0% CP diets, possibly because of greater passage of amino acids to the small intestine. Feeding soybean meal to cows increased production of milk protein compared with feeding corn gluten meal, possibly because more lysine passed to the small intestine.

Amino Acids↗

PubMed1990

Effects of feed processing and frequency of feeding on ruminal fermentation, milk production, and milk composition.

Twenty Holstein cows, averaging 108 d postpartum, were used in five replicated 4 x 4 Latin squares to investigate the effects of feed processing and frequency of feeding on ruminal fermentation, milk production, and milk composition. Four rumen-fistulated cows were used in one of the replicates to monitor ruminal fermentation. Each cow was fed for ad libitum intake a diet of 55% alfalfa and 45% concentrate on a DM basis. Treatments were 1) noncubed diet fed two times daily, 2) noncubed diet fed four times daily, 3) cubed diet fed two times daily, and 4) cubed diet fed four times daily. Alfalfa was fed as long hay in the noncubed diet and chopped and pressed into a cube in the cubed diet. Dry matter intake by cows was not different between treatment comparisons. However, cows fed the noncubed diet consumed 5% more concentrate and 5% less alfalfa than did cows fed the cubed diet. Milk production was greater (1.4 kg/d) when the cubed diet was fed to cows, but the percentage and yield of milk fat were depressed (.43 percentage units and .09 kg/d), causing a decreased production of 4% FCM (.9 kg/d). The depression in milk fat percentage and yield may have been attributed to lowered ruminal fluid pH and a decreased ratio of acetate to propionate in cows consuming the cubed diet. Even though ruminal fluid pH and the ratio of ratio of acetate to propionate tended to be lower when cows were fed four times rather than two times per day, production and composition of milk were not affected by frequency of feeding the diets.

Animal Feed↗

PubMed1990

Effects of feeding alkaline hydrogen peroxide-treated wheat straw-based diets on digestion and production by dairy cows.

Twelve Holstein cows, averaging 34 d postpartum, were used in three replications of a 4 x 4 Latin square design to determine the effects of feeding different levels of alkaline hydrogen peroxide-treated wheat straw on digestion and production responses in lactating dairy cows. Complete mixed diets consisted of 50% concentrate (DM basis) plus varying proportions of treated wheat straw, alfalfa haylage, and corn silage as the forage source. Treatment contained 0 (control), 12.5 (low), 25.0 (medium), or 37.5% (high) treated wheat straw in the diet. Dry matter intakes were 18.5, 17.2, 17.4, and 16.7 kg/d for the four treatments, respectively. Apparent digestibilities of DM and OM were decreased (approximately 4.4 percentage units), and NDF and ADF digestibilities were increased by 9.4 and 3.0 percentage units, respectively, with the high wheat straw diet. Yields of milk and 4% FCM, and SNF percentage did not differ among the treatment groups. Milk fat percentage increased (from 3.07 to 3.32%) and milk protein percentage decreased (from 2.61 to 2.56%) as the proportion of treated wheat straw increased in the diet. Cows fed the higher proportions of treated wheat straw had increased ruminal concentrations of total VFA and molar percentage acetate but a decreased molar percentage propionate, resulting in a greater acetate to propionate ratio. Cows fed the low and medium wheat straw diets had slightly lower DM intakes but production responses were similar to cows fed the control diet containing alfalfa haylage and corn silage as fiber sources.

Animal Feed↗

PubMed1989

Characterization and partial purification of an estrogen type II binding site in chick oviduct cytosol.

An estrogen binding site of moderate affinity (Kd approximately 10 nM) and high capacity (approximately 25-70 pmol/g of tissue) was measured in DES-stimulated chick oviduct cytosol. Saturation analysis by [3H]estradiol exchange demonstrated that this binding site displayed sigmoidal binding characteristics suggesting a cooperative binding mechanism. Competition analysis with a number of compounds demonstrated that the bioflavonoid luteolin was a better competitor for binding to type II sites in chick than either estradiol or DES. Steroid specificity was demonstrated by the inability of 17 alpha-estradiol, progesterone, testosterone, corticosterone, and the triphenylethylene antiestrogen nafoxidine (U-1100A) to compete for [3H]-17 beta-estradiol binding to chick oviduct cytosol preparations. In addition, the binding site appeared to be sensitive to sulfhydryl reducing reagents as evidenced by a 75% reduction in binding activity in the presence of dithiothreitol. Both prelabeling and postlabeling procedures used in conjunction with Sephacryl S-300 chromatography resulted in a single major peak of type II binding activity representing a molecular weight in the 40,000 range. Type II binding activity was recoverable after precipitation with ammonium sulfate, and this material was subjected to a variety of column chromatography procedures in order to achieve further purification of the type II site. Significant purification of the site was achieved with a bioflavonoid-Sepharose (quercetin-Sepharose) affinity matrix. The purified type II sites eluted from quercetin-Sepharose displayed the same sigmoidal binding curves characteristic of native cytosol.

Ammonium Sulfate↗

PubMed1989

Specific binding of estrogen receptor to the estrogen response element.

Gene transfer studies have shown that estrogen regulation of specific genes is mediated by estrogen response elements (ERE). We report that binding of the estrogen receptor to the ERE can be detected by a gel retardation (band shift) assay. This binding interaction was highly sequence and receptor specific. Methylation interference analysis showed that the ERE contact sites of estrogen receptor displayed a perfect twofold rotational symmetry. This is compatible with estrogen receptor binding to the ERE as a head-to-head dimer.

Animals↗