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K Pollow

Publications and source records attributed to K Pollow.

At least 73 records · Page 4Linked to original sources
PubMed1986

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies.

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.

Antibodies↗
PubMed1986

[Monoclonal antibodies for the immunohistochemical detection of estrogen receptor status in tissue sections of primary breast cancers].

The article reports on first experiences with immunohistochemical staining methods (ER-ICA and ER-D5) using monoclonal antibodies for determination of oestrogen receptor status in tissue slides. 95 primary breast cancers were analysed by ER-ICA; in addition, a subgroup of 31 cases was stained for ER-D5. The results of the two immunohistochemical procedures revealed a large measure of agreement with the standard ligand binding assay. The evaluation of the immunohistochemical staining reaction led to the formation of a staining Score: Score points of ER-ICA, not those of ER-D5, revealed good correlation with oestrogen receptor concentrations in cytosol, determined by standard ligand binding assay. The used staining Score was examined for intra- and inter-individual differences of evaluation and proved to be well reproducible. Further clinical studies have to prove how far immunohistochemical features can convey clinically relevant information not yielded by the steroid binding assay.

Antibodies, Monoclonal↗
PubMed1986

Comparison of immunocytochemical estrogen receptor assay, estrogen receptor enzyme immunoassay, and radioligand-labeled estrogen receptor assay in human breast cancer and uterine tissue.

Determination of estrogen receptor content in 82 breast cancer specimens with immunocytochemical estrogen receptor assay (ER-EIA) (Abbott) was compared with our routinely used binding assay using 125I-estradiol as radioligand with Scatchard plot analysis of the binding data. Although the estrogen receptor content measured with the ER-EIA was approximately 2-fold higher compared with the binding assay, the immunochemical method proved to be a useful alternative for estrogen receptor determination. Furthermore, it is possible to detect estrogen receptors in FPLC Superose 12 (size exclusion column) eluates or in the fractions obtained after sucrose density centrifugation using the ER-EIA. Forty breast cancer samples were analyzed utilizing the immunocytochemical technique (ER-ICA) for visualization of the estrogen receptor content in frozen tumor tissues in relationship to the quantitative results obtained with the ER-EIA assay. Specific staining for estrogen receptor was confined only to the cell nucleus, was distributed irregularly among the tumor cells, and was variable in intensity. The staining intensity and the percentage of positively stained cells increased with increasing level of cytosolic estrogen receptor. In 27 of 40 cases the immunocytochemical results correlated well with the ER-EIA assay. Nine cases were ER-ICA negative with positive ER-EIA, and four were ER-ICA positive with negative ER-EIA.

Breast Neoplasms↗
PubMed1985

3H-cyproterone acetate: binding characteristics to human uterine progestagen receptors.

The availability of tritium labeled cyproterone acetate (CPA) facilitated the systematic investigation of the binding characteristics of this compound for human uterine progesterone receptors (PgR). The binding parameters of 3H-CPA are compared to those of 3H-R5020 and 3H-progesterone. The rate constants of association (k1M-1sec-1) to PgR were 7.8 X 10(3) for 3H-R5020, 4.5 X 10(4) for 3H-progesterone and 4.0 X 10(4) for 3H-CPA. The rate constants of dissociation (k-1, sec-1) were 3.6 X 10(-5) for 3H-R5020, 21.3 X 10(-5) for 3H-progesterone and 17.8 X 10(-5) for 3H-CPA. The Kd-values (M), as obtained by titration analysis and subsequent Scatchard plot analysis were 1.2 X 10(-9) for 3H-R5020, 6.0 X 10(-9) for 3H-progesterone and 5.2 X 10(-9) for 3H-CPA. On sucrose density gradient analysis binding in the 3.5, 5 and 8 S area could be observed using 3H-R5020. For 3H-progesterone and 3H-CPA binding was exclusively found in the 5 S area. The specificity of the steroid binding site of PgR is identical for 3H-R5020 and 3H-CPA. The order of potency of binding for various competitors decreases identically for both radioactive ligands: R5020 less than progesterone less than R1881 less than CPA less than dihydrotestosterone less than dexamethasone less than cyproterone less than 15 beta-OH-cyproterone. CPA resembles progesterone very closely in its binding characteristics to human uterine PgR.

Binding, Competitive↗
PubMed1985

Immunohistochemical demonstration of carcinoembryonic antigen (CEA) in 120 mammary carcinomas and its correlation with tumor type, grading, staging plasma-CEA, and biochemical receptor status.

Antisera to CEA were used for the immunohistochemical localization and quantification of this antigen in 120 Bouin-fixed, paraffin embedded mammary carcinomas. These results were compared to tumor type, grading, staging, biochemical receptor status, cytosolic CEA-levels of the same tumors, and preoperative plasma CEA-levels. Mammary carcinomas were usually characterized by a low percentage of CEA-positive tumor cells: 50.9% of the cases contained more than 5% CEA-positive tumor cells and were therefore defined as being CEA-histopositive in this study. A relation could be shown between CEA-histopositivity and the histologic tumor type. The majority of invasive lobular carcinomas, tubular, and cribriform carcinomas was CEA-negative (72%). Conversely, 70% of invasive ductal carcinomas were CEA-positive. There was a significantly higher percentage of CEA-histopositivity in grade III tumors than in grade I/II carcinomas. The results obtained by quantification of the immunohistochemical staining of CEA were positively correlated with the results obtained by cytosolic CEA-assay. The overall concordance between tissue and plasma determinations of CEA was found to be 57.1%. A positive trend could be found between CEA-positivity and staging. However, no correlation was observed between CEA-positivity and estrogen receptor status.

Adult↗
PubMed1985

A simple, low-cost computer program for Scatchard plot analysis of binding data in steroid hormone receptor assays.

This paper presents a relatively simple computer program for Scatchard plot analysis of binding data in steroid hormone receptor determination. The program is written in BASIC and adapted to a low-cost desktop calculator. The program is designed to permit data input without demanding extensive technical knowledge of either computer systems or mathematical background of receptor binding assays.

Breast Neoplasms↗
PubMed1985

Characterization of a high-affinity, low-capacity androgen binding protein in human term placental cytosol.

A high-affinity, low-capacity androgen binding component in human placental cytosol was characterized. (3H)-R1881, a synthetic androgen, was used as radioactive ligand for characterization and quantification of this placental androgen binding component. Various naturally occurring as well as synthetic steroids were first investigated for their in vitro stability under receptor assay conditions (i.e., an overnight incubation at 4 degrees C). Only the synthetic steroids were found to be metabolically stable. The rate constant of dissociation was found to be 6.42 X 10(-4) s-1 (on the basis of k-1 the rate constant of association was 1.27 X 10(5) M-1 s-1). The concentration of androgen binding protein was measured in 50 human term placentae. The mean concentration was 169 +/- 132 fmol/mg protein, and the average Kd was 5.07 +/- 2.2 X 10(-9) M. Placentae of female fetuses contained significantly higher concentrations (219 +/- 140 fmoles/mg protein) than those of male fetuses (115.2 +/- 101 fmoles/mg protein). The (3H)-R1881-androgen binding protein complex exhibited a sedimentation coefficient of 4S under low salt conditions. The binding site was only specific for androgens and progestins but not for steroids of other classes.

Androgen-Binding Protein↗
PubMed1985

Oral high-dose medroxyprogesterone acetate (MPA) treatment: cortisol/MPA serum profiles in relation to breast cancer regression.

Medroxyprogesterone acetate (MPA), a potent synthetic progestin, has been widely used in the hormonal treatment of advanced breast cancer, but presently with varying dose schedules. The availability of a sensitive RIA-method for determination of serum MPA has stimulated the research on MPA serum levels in patients after repeated MPA administration. The aim of this study was to assay blood level profiles of MPA as well as of cortisol during repeated high-dose orally administered MPA. 34 patients with metastatic breast cancer were enrolled in this study. 12 patients died already within the first 4 weeks of MPA treatment due to multiple metastases. The dosage regimen based on the daily oral administration of 1,000 mg MPA suspension. During MPA treatment, a decrease of cortisol serum levels was observed in nearly all patients. Within the observation time of 8 months out of 22 evaluable cases 18.2% responded to the therapy (complete and partial response). No change was observed in 36.4% and progression in 45.5% of the patients. Within the remission (complete and partial) group, a good correlation between constant MPA serum levels above 150 ng/ml and remission was observed. But in the groups with no change and progression no such correlation could be observed.

Administration, Oral↗
PubMed1985

Radioimmunoassay of human serum serotonin.

A radioimmunoassay for extracted, N-acetylated human serum serotonin (5-hydroxytryptamine) is described. Antisera were raised in rabbits against a conjugate of bovine serum albumin with serotonin hemisuccinamide. Polyethylene glycol in combination with anti-rabbit immunoglobulins was used to separate bound and unbound 125I-Bolton Hunter-serotonin conjugate. Ethanol precipitation of serum proteins was used to extract serotonin, which was subsequently acetylated with acetic anhydride to N-acetyl serotonin. The average recovery was 66%. The minimal detectable concentration of N-acetyl serotonin was 0.012 mumol/l serum (25 fmol per tube). The intra-assay precision (CV) was 6.8% (n = 20) at a level of 0.9 +/- 0.06 mumol/l. The inter-assay CV was 10% at a level of 0.49 +/- 0.049 mumol/l, and 25% (n = 10) at a level of 2.16 +/- 0.53. Analytical recovery of serotonin, corrected for losses during extraction and acetylation, was 99 +/- 13%. The only substance cross-reacting with the antibody was endogenous N-acetyl serotonin. This was detectable when the acetylation step was omitted, and it can be removed by extraction before the acetylation. The observed range for the concentration of serotonin in serum was for 59 women 0.45 - 3.46 (mean +/- SD: 1.37 +/- 0.63 mumol/l) and for 59 men 0.19 - 2.8 (mean +/- SD: 1.18 +/- 0.56 mumol/l). All values are corrected for endogenous N-acetyl serotonin: observed range 0 - 0.18 (mean +/- SD: 0.03 +/- 0.03 mumol/l).

Adolescent↗
PubMed1985

3H-labelled RU 38486: characterization of binding sites in human uterine cytosol.

The behaviour of the antifertilizing synthetic steroid RU 38486 towards human uterine progestin receptor was investigated. RU 38486 competed in the same order of magnitude as progesterone for the [3H]R 5020 binding site of progestin receptor, whereas R 5020 was unable to compete against [3H]RU 38486. This apparent contradiction could be explained by means of HPLC-chromatography. HPLC-chromatography with an anion exchange column (MonoQ, Pharmacia, Uppsala, Sweden) showed that [3H]RU 38486 forms at least two stable complexes with uterine cytosol, on one hand with serum albumin, which presents almost 90% of bound radioactivity, and on the other hand with the two native progestin receptor forms, corresponding to 4 S and 8 S receptor forms in sucrose density gradient analysis. Whether reduced binding of salt-activated RU 38486 receptor complexes to DNA-cellulose is due to reduced activation is still uncertain and remains to be further investigated.

Abortifacient Agents↗
PubMed1984

High-performance and ion-exchange chromatography and chromatofocusing of the human uterine progesterone receptor: its application to the identification of 21-[3H]dehydro Org 2058-labelled receptor.

Two independent lines of evidence were used to identify the human uterine progesterone receptor. First, three differently tritiated progestogens (Org 2058, R 5020, progesterone) were used for reversible labelling of the receptor. Secondly, the highly potent affinity label 21-[3H]dehydro Org 2058 was used to label covalently the steroid-specific binding site of the receptor. The labelled cytosols were chromatographed on a Mono Q high-performance anion-exchange column in the absence or presence of a high molar excess of the respective unlabelled competitor steroids. In the case of 21-[3H]dehydro Org 2058, Org 2058 was used as the unlabelled competitor. After elution with a NaCl gradient, the radioactivity was determined in each fraction and the elution profiles (absorption, A at 280 nm; radioactivity, dpm) were superimposed. Free steroid was eluted with the washing buffer. When the NaCl gradient was performed, two peaks of radioactivity were located. The specifically protein-bound radioactivity was eluted at 0.08 M NaCl. Two non-specific steroid-binding entities were eluted at 0.1 and 0.22 M NaCl, the second of which was identified as albumin. The elution profiles of tritiated progesterone, R 5020, Org 2058 and the affinity label 21-dehydro Org 2058 were identical. In a second set of experiments, Org 2058- and 21-dehydro Org 2058-labelled cytosols were subjected to high-performance liquid chromatography on a Mono P high-performance chromatofocusing column in the absence or presence of a high molar excess of unlabelled Org 2058. After elution with Polybuffer 74, only one specifically labelled protein (pH 6.4) was detected. When the Mono P-purified receptor was submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis, two labelled polypeptides with Mr = 45,000 and 27,000 were detectable.

Binding, Competitive↗
PubMed1984

Peanut lectin histochemistry of 120 mammary carcinomas and its relation to tumor type, grading, staging, and receptor status.

Peanut lectin (PNL) is known to bind beta-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which provides antigenic determination of the Thomsen-Friedenreich antigen ( TFAg ). The aim of this study was to analyse the expression of peanut lectin binding sites in mammary carcinomas and to correlate these with tumor type, histological grading, staging and biochemical receptor status. The series comprised 120 invasive mammary carcinomas and 14 cases with normal breast tissue or benign epithelial proliferations as controls. In controls mainly luminal or apical PNL-binding was discovered, however, in all except three carcinomas a cytoplasmatic localisation of TFAg with three major patterns was found: diffuse, granular-globular and vacuolar reactions. The quantitative-qualitative evaluation of the PNL-staining revealed a statistically significant correlation between globular-vacuolar PNL-reaction and tumor type with a higher percentage of this type of reaction in invasive lobular carcinomas as opposed to tubular and invasive ductal carcinomas. Furthermore a statistically significant relationship was disclosed between PNL- histopositivity and estrogen positive - progesterone positive cases. However, the findings of contradictory PNL-status and hormone-receptor status illustrates clearly the difficulty of predicting the biochemical receptor status. No correlation was found between PNL-histochemistry, histological grading, and pathological staging. The practical implications of PNL-histochemistry of mammary carcinomas are discussed.

Arachis↗
PubMed1984

Characterization of human spleen tumor glucocorticoid receptors using [3H]cortisol as ligand.

The present report describes an assay system allowing the quantification and characterization of [3H]cortisol binding to glucocorticoid receptors in bloodrich human tissue. The essence of this assay lies in the selective binding of 17 beta-carboxylic acids of natural corticoids to corticosteroid binding globulin (CBG). In the presence of 1240 nmol/l 11 beta-hydroxy-3-oxo-4-androstene-17 beta-carboxylic acid only glucocorticoid receptors were detectable with the expected properties: high affinity for synthetic and natural glucocorticoids, but failure to bind to the respective 17 beta-carboxylic acids, apparent Kd's at 0-4 degrees C for [3H]cortisol of approx 30 nmol/l, Nmax similar to those determined with [3H]dexamethasone and the typical sequence of relative binding affinities (dexamethasone greater than cortisol greater than progesterone greater than 17 beta-methyl-testosterone, estradiol).

Binding, Competitive↗
PubMed1984

[A new LH-RIA rapid test for determining peak levels of serum LH--a methodological contribution to in-vitro fertilization].

A highly sensitive, easy to operate LH-RIA rapid test was developed by changing the incubation period and temperature and by reducing the standard curve to three measurement data, using commercially available reagents. The results obtained by means of the LH-RIA rapid test correlate most loosely with those of a conventional 5-hour LH-RIA. The new LH-RIA rapid test is ideally suited for LH peak search at short intervals for exact determination of the time of ovulation as part of in-vitro fertilisation or insemination.

Adult↗
PubMed1984

Criteria for the establishment of a double-labeling assay for simultaneous determination of estrogen and progesterone receptors.

The availability of [125I]-16 alpha-iodo-3,17 beta-estradiol ( [125I]-E2) with binding characteristics similar to estrogen receptor (ER) enabled us to establish a double-labeling assay for the simultaneous determination of ER and progesterone receptors (PgR) using 125I-E2 and [3H]-R5020. The criteria for the establishment of such a double-labeling assay are described. 150 human mammary tumor cytosols have been investigated with the standard routine receptor assay for ER as well as PgR and the results were compared to those obtained by the double-labeling assay. ER: a coefficient of correlation of 0.691 was obtained, the parameters of the regression line were Y = 1.025 X X-0.036. When referring to the standard assay, 3 determinations were false-positive and 4 false-negative. PgR: a correlation coefficient of 0.984 was found, the parameters of the regression line were Y = 0.960 X X + 12.16. One value was false-negative and 1 false-positive. An equivalence of the two methods could be demonstrated. This new assay reduces by half the amount of tissue necessary for a valid 4- to 6-point saturation analysis, the time required for performing the assay and its cost.

Binding, Competitive↗
PubMed1984

17 beta-carboxamide steroids: highly effective inhibitors of the phytohaemagglutinin mediated blastogenesis of normal human peripheral lymphocytes.

Several novel 17 beta-carboxamide analogues of dexamethasone were synthesized. The common precursor, 9-fluoro-16 alpha-methyl-11 beta,17-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxylic acid, did not bind to the glucocorticoid receptors of rat liver and human spleen tumours. In addition, no inhibition of the mitogen-induced blastogenesis of cultured human peripheral lymphocytes was observed. The 17 beta-carboxamide analogues, however, bound with similar affinities to the glucocorticoid receptors of both tissues. They inhibited the mitogen-induced blastogenesis of peripheral lymphocytes, showing the same potency and same order of binding affinity as the natural glucocorticoids.

Animals↗
PubMed1983

3H-estradiol and 3H-R5020 binding in cytosols of normal and neoplastic human ovarian tissue.

High-affinity cytoplasmic estrogen and progesterone receptors in normal and abnormal ovarian tissues were studied. Estradiol receptor was detectable in 65% and progesterone receptor in 36% of the malignant tumors; 39% of all malignant ovarian tissues were estradiol- as well as progesterone-receptor-positive. Tumors were said to be receptor-positive when the receptors bound greater than 5 fM steroid/mg cytosol protein. No correlations were found between receptor status and histopathological diagnosis. In normal ovarian tissues collected at various phases of the menstrual cycle no changes in [3H]-estradiol and [3H]-R5020 binding could be detected. Analysis of the receptor concentration for both steroid hormones with regard to the menopausal status demonstrated highest levels in postmenopausal women. No significant difference could be found when two groups of patients with advanced ovarian carcinoma associated with the cytosol estrogen receptor status were compared in terms of two different therapeutic schemes (cytosolic chemotherapy with and without tamoxifen).

Cytosol↗