Demonstration of luteal cell membrane receptors for prostaglandin F2 alpha by ultrastructural and binding analysis.
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Biomedical subjects
Publications and source records attributed to K Wright.
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The urinary excretion of estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate in men and throughout the menstrual cycle in women was measured by specific radioimmunoassay. In 9 men the mean +/- SE excretion of these conjugates was 15.9 +/- 1.4, 2.7 +/- 0.3, and 3.2 +/- 0.2 microgram/24 h respectively. In 15 women studied in the midfollicular phase (day 8) of the menstrual cycle, the excretion was 19.4 +/- 1.7, 2.9 +/- 0.2, and 5.4 +/- 1.3 micrograms/24 h. Excretion of each conjugate was significantly (P less than 0.01) elevated in the midluteal phase (day 22) to 41.9 +/- 3.9, 6.3 +/- 0.8, and 12.2 +/- 1.5 micrograms/24 h respectively (n = 14). The mean excretion of estriol-16 alpha-glucosiduronate was greater than that of 17 beta-estradiol-17-glucosiduronate in the luteal phase (P less than 0.05) but not in the follicular phase or in men (P greater than 0.05). The excretion of each of these specific conjugates measured throughout the menstrual cycle in 7 women was characterized by a sharp midcycle peak and a lower, broader luteal phase peak. The ratios of estriol-16 alpha-glucosiduronate to 17 beta-estradiol-17-glucosiduronate, estrone glucosiduronate to 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate to estrone glucosiduronate throughout the menstrual cycle were analyzed. When the mean ratio during the follicular phase was set at 1, a significant increase (P less than 0.01) occurred in the mean luteal phase ratio in each case: 1.00 +/- 0.03 to 1.66 +/- 0.09, 1.00 +/- 0.04 to 1.30 +/- 0.04, and 1.00 +/- 0.03 to 1.24 +/- 0.04 (mean +/- SE) respectively. The marked alteration in the proportions of these urinary estrogen conjugates may be due to altered metabolism of 17 beta-estradiol, but it more likely reflects a change in the pattern of estrogen secretion or production between the two phases of the menstrual cycle.
Prostaglandin F2 alpha (PGF2 alpha) binds specifically to a partially purified membrane preparation from rat corpora lutea. The high affinity, low capacity binding component asa a Kd = 4.7 nM and has a capacity of 0.38 pmol/mg protein. Binding kinetics were temperature-dependent with an association rate constant of 2.5 x 10(5) 1/mol-sec and a dissociation rate constant of 4.3 x 10(-4) sec-1 at 22 degrees C. Little competition for binding was shown by other prostaglandins and prostaglandin metabolites; the PGF2 alpha analogue ICI 81008 (16-m-trifluoromethylphenyl-prostaglandin F2 alpha) showed a binding affinity similar to that of PGF2 alpha. The specific binding of PGF2 alpha to luteal cell membranes was confirmed by electron microscopy using a ferritin--PGF2 alpha conjugate. Ferritin--PGF2 alpha was found predominantly on luteal cell surfaces; little binding occurred on other types of cells present. These data demonstrate specific binding of PGF2 alha to rat luteal membranes. It is suggested that the luteolytic action of PGF2 alpha in the rat may be receptor-mediated.
The luteolytic action of PGF2 alpha appears to be due to loss of gonadotropin support of corpus luteum function. This "chemical hypophysectomy" produced by PGF2 alpha in the rat has several components: 1. A rapid block of gonadotropin uptake in vivo by an unexplained mechanism. 2. A rapid and direct block of adenylate cyclase activation by the LH receptor complex in isolated luteal cells. 3. An eventual loss of LH receptor binding activity produced by inhibition of prolactin action.
A direct radioimmunoassay which does not require hydrolysis or chromatography has been developed for estriol-16alpha-glucosiduronate in pregnancy plasma and urine. The antigen used in the development of this antiserum was prepared by joining the carboxylic acid group of estriol-16alpha-glucosiduronate covalently to the epsilon-amino group of the lysine residues in bovine serum albumin. Results indicate that the direct radioimmunoassay yields levels of estriol-16alpha-glucosiduronate in late pregnancy urine that are comparable to those obtained by mroe elaborate procedures involving chromatographic separation of the estriol conjugates, followed by enzymic hydrolysis and measurement of the freed estriol. In addition, good correlation was found between the total estrogen values in urine obtained by currently used chemical procedures and urinary levels of estriol-16alpha-glucosiduronate. The renal clearance of estriol-16alpha-glucosiduronate was determined in eight normal women during the third trimester, and the mean value +/- S.D. was 404 +/- 81 ml. per minute (range, 248 to 494). This method is suitable for the evaluation of variations in the plasma and urine levels of estriol-16alpha-glucosiduronate in pregnancy and offers significant advantages over the presently used chemical methods for monitoring fetal well-being.
Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.
This article describes an exercise which was designed by a multidisciplinary group, including a community physician, health economist, clinician and operational research practitioner, to explore the essential nature of planning services for a particular health care group. The first programme, described in the article, was concerned with psychogeriatric services, including both community and hospital provision, but it is indicated that the exercise can be adapted to all other services, and has already been used to evolve a comprehensive accident and emergency service in a particular health area. Participants came from a wide variety of disciplines, including community medicine, psychiatry, geriatrics, hospital and community nursing and non-medical administration. The tutors to the course were those who had been involved in its planning and they spent the time working with the participants to explore the concepts involved and to produce some practical solutions that could be used in the field.
Specific RIAs requiring ether extraction only were established for estrone and 17 beta-estradiol both in plasma and in urine from the nonpregnant female. These assays were used to measure the renal clearance rates of estrone and of 17 beta-estradiol in eight ambulatory women in the follicular and in the luteal phases of the menstrual cycle. The mean (+/-SE) for the renal clearance rate of estrone was 0.71 +/- 0.058 ml/min in the follicular phase and 1.26 +/- 0.35 ml/min in the luteal phase. The mean (+/-SE) renal clearance rate of 17 beta-estradiol was 0.44 +/- 0.055 ml/min in the follicular phase and 0.29 +/- 0.043 ml/min in the luteal phase. There was no significant difference in the renal clearance rates of either estrone or of 17 beta-estradiol between the follicular and luteal phases of the cycle. The renal clearances of estrone and 17 beta-estradiol were highly correlated (r = 0.84; P less than 0.01). The renal clearance rate of estrone was significantly greater than that of 17 beta-estradiol in both phases of the cycle (P less than 0.01).
Estrone sulfate, quantitatively the most important estrogen in plasma, has previously been determined only after hydrolysis and chromatography. An antiserum raised against estrone glucosiduronate-bovine thyroglobulin was found to be suitable for the specific RIA of estrone sulfate both in plasma and urine. Plasma levels were measured after solvent extraction without hydrolysis or chromatography. The mean (+/-SE) was 972 +/- 79 pg/ml (range, 537-1581) in 15 women in the follicular phase, 1806 +/- 160 pg/ml (range, 814-3358) in 15 women in the luteal phase, and 922 +/- 62 pg/ml (range, 461-1238) in 13 men. The urinary excretion of estrone sulfate, measured after simple chromatographic separation, ranged from 0.8-7.9 micrograms/24 h in men and 5.1-18.7 micrograms/24h in nonpregnant women. This was generally one-seventh to one-half the simultaneous estrone glucosiduronate excretion rate. An approximate mean renal clearance of estrone sulfate calculated from the above values was 2.7 ml/min. The low clearance rate is taken to reflect extensive binding of estrone sulfate by plasma proteins. The splanchnic extraction of estrone sulfate measured in 6 patients undergoing hepatic vein catheterization for diagnostic purposes was 29.8 +/- 11.1%, indicating net uptake of this compound by the splanchnic area.
The distance between aspartokinase and homoserine dehydrogenase active sites was determined using fluorescence energy transfer between modified substrates. The fluorescent 1,N(6)-ethenoadenosine 5'-triphosphate was bound at the kinase active site by Co(III) affinity labeling. Reduced thionicotinamide adenine dinucleotide phosphate quenched the fluorescence of bound nucleotide. Fluorescence depolarization measurements led to a delimitation of the value of the dipolar orientation factor to the range 0.3 to 2.8. The distance between the fluorescent probe and the quencher was 29 +/- 4 A. In the presence of threonine, this distance increased to 36 +/- 5 A. Threonine binding either increased the intersite distance by ca. 7 A or caused a reorientation of the probe at the dehydrogenase site.
The nature of the feedback inhibition of the bifunctional enzyme, aspartokinase I-homoserine dehydrogenase I of Escherichia coli was studied using 13C nuclear magnetic resonance (NMR). Since aspartokinase is activated by Mn(II), the interaction of the inhibitor L-threonine (specifically enriched to 90% 13C in the carboxyl carbon) with the metal-enzyme complex was studied. Spin-lattice (T1) and spin-spin (T2) relaxation times were determined by the partially relaxed Fourier transform method and line-width measurements respectively at 20 MHz. The pronounced broadening of the DL-threonine carboxyl peak in the presence of the Mn(II)-enzyme complex indicates that an L-threonine binding site is close to the metal binding site of the kinase active site. The non-identity of (T1)*M and (T2)*M indicates that conditions of fast exchange prevail. The (T1)*M/(T2)*M ratio was used to estimate a correlation time of 2.0 ns for the dipolar interaction at 25 degrees C. An estimate for the distance between Mn(II) and the threonine carboxyl carbon of 4.4 A (0.44 nm) was obtained. This 13C NMR study has thus located one of the two classes of threonine regulatory sites which exist per subunit; the threonine site identified here is at the aspartokinase active site, adjacent to the catalytic metal site.
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A reproduceable method has been developed for measuring the quality of clinical care provided by physicians and nurse practitioners. The distinctive features of the method are the extended use of the tracer disease concepts, the evaluation of referrals, new procedures for probing the clinical operation of practices, a single blind design, emphasis on the use of the untouched medical record, the ability to compare results with measurements of concurrent outcome, and a relatively low cost. Three simultaneous approaches used in the method are described: surveillance of the management of indicator conditions, evaluation of clinical use drugs, and the assessment of referral decisions. The three approaches gave consistently similar results about the relative performances of the practices compared and were in agreement with concurrent outcome studies. The method was successfully implemented in a health care experiment.
1. The patterns of incorporation of radioactivity from d-[U-(14)C]glucose into the pectic components of sections of sycamore roots changed so that sections nearer the tip incorporated relatively more label into arabinose and galactose compared with uronic acid. 2. Radioactive maize root-cap slime was prepared and found to contain three water-soluble component polymers which were electrophoretically (i) neutral, (ii) weakly acidic and (iii) strongly acidic at pH6.5. The neutral component was a glucan. The other components, which could be degraded by trans-elimination, consisted of an acidic backbone chain composed of galacturonic acid and glucose, attached to which were different proportions of neutral sugars. Arabinose, galactose and fucose, the main neutral sugars of the weakly and strongly acidic materials, were absent from the neutral fraction. 3. Fucose was a major sugar in maize-root slime and in a slime of similar composition synthesized by a maize callus of shoot origin. Only trace amounts were found in sycamore, pea and wheat root tips, and in pectin prepared from maize roots and coleoptiles. A high proportion of fucose is therefore a chemical characteristic of maize slime, and slime synthesis indicated a state of differentiation of the tissue. 4. The similarity between the slime and pectin is discussed; slime is a form of pectin modified in such a way as to provide a hydrated protective coating around the root tip.
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