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Biomedical subjects

E Mulder

Publications and source records attributed to E Mulder.

At least 55 records · Page 3Linked to original sources
PubMed1989

Actively replicating nucleoids influence positioning of division sites in Escherichia coli filaments forming cells lacking DNA.

The positioning of constrictions in Escherichia coli filaments pinching off anucleate cells was analyzed by fluorescence microscopy of dnaX(Ts), dnaX(Ts) sfiA, dnaA46(Ts), gyrA(Am) supF(Ts), and gyrB(Ts) mutants. In filaments with actively replicating nucleoids, constrictions were positioned close to the nucleoid, whereas in nonreplicating filaments, positioning of constrictions within the anucleate region was nearly random. We conclude that constriction positioning depends in an unknown way on nucleoid replication activity.

Cell Division↗
PubMed1989

Androgen receptor-mediated growth and epidermal growth factor receptor induction in the human prostate cell line LNCaP.

Human prostate tumor cells, LNCaP, contain androgen receptors and respond to androgens with increased growth rate. Although other steroid hormone receptors are not detectable in LNCaP cells, progesterone and estradiol stimulate the growth of these cells. The androgen receptor shows considerable cross binding activity with progesterone and estradiol but not with the glucocorticoid triamcinolone acetonide. The latter steroid does not have any effect on cell proliferation. LNCaP cells respond to epidermal growth factor (EGF) with an increased growth rate. Steroids that stimulate LNCaP cell proliferation also increase the number of EGF receptors per cell. In conclusion, LNCaP cells are sensitive to EGF. Different steroids bind to the androgen receptor and stimulate the proliferation of human prostate tumor cells. This stimulation of growth is preceded by an increase in EGF receptor number per cell.

Androgens↗
PubMed1988

Regulation of growth and epidermal growth factor receptor levels of LNCaP prostate tumor cells by different steroids.

The growth of LNCaP cells, derived from a lymph-node carcinoma of the human prostate, was stimulated by different hormones. Optimal growth (3- to 4-fold increase in DNA content per culture versus controls) was observed at 0.1 nM R1881 (a synthetic androgen), 1 nM progesterone or 10 nM estradiol. Triamcinolone acetonide had no effect. Dihydrotestosterone maximally stimulated cell growth at 10 nM. When the culture medium was changed 4 times in 6 days instead of twice, optimal growth was observed at 1 nM dihydrotestosterone. This indicates that a rapid metabolism of dihydrotestosterone influenced growth response. LNCaP cells contained considerable amounts of androgen receptors (920 fmol/mg cytosol protein) while progestagen, estrogen and glucocorticoid receptors were absent. The affinity of steroids for the androgen receptor decreased in the order of: R1881 (relative binding affinity: 100.0) greater than dihydrotestosterone (67.7) greater than progesterone (29.4) greater than testosterone (23.8) greater than estradiol (4.3) greater than triamcinolone acetonide (less than 0.1). Effects on cell growth of these steroids paralleled their affinity for the androgen receptor. The number of EGF receptors per cell increased in a dose-dependent manner upon treatment with various hormones. Again the amount of steroid needed for maximal effects reflected the affinity of the steroid for the androgen receptor. An approximately 2-fold increase in EGF receptor number was observed within 24 hr and before an increase in growth could be detected. Actinomycin-D and cycloheximide inhibited the hormonally induced increase in EGF receptor numbers.

ErbB Receptors↗
PubMed1988

Purification of the intact monomeric 110 kDa form of the androgen receptor from calf uterus to near homogeneity.

In the present study, calf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl2. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [3H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5 while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [3H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa.

Affinity Labels↗
PubMed1988

Cloning, structure and expression of a cDNA encoding the human androgen receptor.

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.

Amino Acid Sequence↗
PubMed1988

The variable region of the Trypanosoma brucei kinetoplast maxicircle: sequence and transcript analysis of a repetitive and a non-repetitive fragment.

The sequence of two fragments derived from the variable region of the kinetoplast maxicircle of Trypanosoma brucei has been determined. One fragment (1334 nucleotides, situated immediately upstream of the 12S and 9S ribosomal RNA genes) consists of non-repetitious DNA, which does not hybridize to other maxicircle regions. The other (844 nt, located between 1.7 and 2.55 kb downstream of the NADH-dehydrogenase subunit 5 gene) contains arrays of repetitive sequences which are also found outside this area. Hybridization analysis suggests that approximately 60% of the remaining part of the divergent region, which has not yet been fully analyzed, consists of similar sequences. Neither segment contains genes for mitochondrial proteins or tRNAs, as judged from computer analysis. This conclusion is supported by the fact that maxicircle DNA of trypanosome species other than T. brucei does not cross-hybridize to either fragment. Northern blot analysis and S1 nuclease experiments demonstrate, however, that both maxicircle regions are transcribed into RNAs of varying length (100-3000 nt), albeit at a low level. The function of these transcripts, that are derived from both DNA strands, and the likely absence of protein and tRNA genes from the variable region of the T. brucei maxicircle is discussed.

Animals↗
PubMed1988

Androgens stimulate both growth rate and epidermal growth factor receptor activity of the human prostate tumor cell LNCaP.

LNCaP cells (derived from a lymph node carcinoma of the human prostate) contain androgen receptors and show androgen-responsive growth in vitro. Maximal effects on growth were seen at 0.1 nM of the synthetic androgen R1881 or 0.2 nM of epidermal growth factor (EGF); both compounds independently increased the growth rate 2-3 times. EGF-receptors were measured after 6 days culture in the presence or absence of 0.1 nM R1881. A 2.3-fold increase in receptor number/cell was found when binding was measured at 0 degrees C (from 12,500 to 28,900 sites/cell in stimulated cells). The kD value (0.45 nM) was not affected by androgen treatment. The increase of EGF-receptor activity was first observed between 6 and 12 h after exposure to androgen. It is concluded that LNCaP cells are sensitive to low concentrations of EGF (or EGF-like compounds) and that one of the mechanisms involved in androgen action on these cells is an increase of EGF-receptor expression at the cell surface.

Androgens↗
PubMed1988

In situ photolabelling of the human androgen receptor.

In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.

Affinity Labels↗
PubMed1988

Androgens and transforming growth factor beta modulate the growth response to epidermal growth factor in human prostatic tumor cells (LNCaP).

UNLABELLED: Treatment of LNCaP human prostatic cancer cells with 0.1 nM of the synthetic androgen, R1881, resulted in a three-fold stimulation of growth in 6 days. Of several growth factors tested (epidermal growth factor (EGF), platelet-derived growth factor, insulin-like growth factor, and insulin) only EGF (1 ng/ml) stimulated cell growth (two-fold). This stimulatory effect of EGF was inhibited for approximately 70% by 0.02 ng transforming growth factor beta (TGF beta)/ml. EGF (1 ng/ml) acted synergistically with R1881 (0.1 nM) on LNCaP cells to induce cell proliferation (seven-fold increase in cell growth). The synergistic effect of androgen and EGF was inhibited by TGF beta (0.05 ng/ml). IN CONCLUSION: human prostatic LNCaP cells are sensitive to EGF. Androgen increases and TGF beta decreases the growth response to EGF. This effect of TGF beta on an androgen-responsive system has not been observed before.

Androgens↗
PubMed1988

Treatment of bulimia with fenfluramine and desipramine.

Desipramine and fenfluramine were administered to bulimic patients in a 15-week study of double-blind, placebo-controlled, crossover design. The 22 patients in the study met DSM-III criteria for bulimia and were of normal weight. Twelve subjects were randomly allocated to the fenfluramine group, and 10 subjects received desipramine. Half the subjects in each group received the active drug in the first 6 weeks and half received placebo. There was a 3-week washout period, after which subjects were crossed over for the remaining 6 weeks. The Eating Disorder Inventory, profile of Mood States, bulimia symptom checklists, and Hopkins Symptom Checklist were administered at weeks 0, 2, 4, 6, 9, 11, 13, and 15. Subjects maintained a daily record of bingeing, vomiting, and laxative/diuretic abuse. Results indicated that both drugs had beneficial effects on bingeing and vomiting frequency, although a greater proportion of patients were identified who responded to fenfluramine than to desipramine. Fenfluramine and desipramine were also effective in reducing the psychological symptoms of bulimia, such as the urge to binge, and feelings of depression. Results suggest that direct alteration of central food intake regulatory centers can effectively control bulimia.

Adolescent↗
PubMed1987

Characterization of androgen receptors in a transplantable human prostatic adenocarcinoma (PC-82).

The transplantable human prostatic adenocarcinoma, PC-82, has been shown to be a suitable model for the study of several aspects of androgen-regulated tumor growth. This tumor contains an androgen receptor, and the purpose of the present investigation was to characterize this androgen receptor with respect to hormone specificity, sedimentation coefficient, dissociation constant, Stokes radius, ionic properties, and molecular mass. Cytosol was prepared from tumor tissues grown in athymic nude mice, which were castrated 10 days before harvesting the tumor. Scatchard plot analysis revealed a binding protein with a Kd of 0.1 nM for R1881 (methyltrienolone) and binding capacity of 120 fmol/mg protein. The receptor showed a high affinity for R1881, testosterone, and 5 alpha-dihydrotestosterone, respectively, whereas no or little affinity was found for progesterone and estradiol. In the presence of 10 mM molybdate the androgen receptor in PC-82 cytosol eluted from an FPLC anion exchange column (Mono Q) at 0.32 M NaCl, which is identical to what has been found for androgen receptors from rat prostate and calf uterine cytosol. Photoaffinity labeling of the [3H]R1881-androgen receptor complex and subsequent analysis on SDS-polyacrylamide gels resulted in a covalently labeled protein with a molecular mass of approximately 50 kD. The androgen receptor of the PC-82 tumor had a sedimentation coefficient of 4S and a Stokes radius of 3.3 nm at high ionic strength (0.4 M NaCl). It is concluded that the PC-82 tumor contains a binding protein with the properties described for androgen receptors present in prostate tissue.

Adenocarcinoma↗
PubMed1987

Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis.

The DNA-binding form of the calf uterine androgen receptor (AR) was subjected to limited protease digestion using chymotrypsin, trypsin and a rat prostate cytosol protease. The properties of the generated polypeptide fragments were identified and compared with those of the intact AR. Physicochemical characterization was achieved through sedimentation analysis, gel filtration chromatography and DEAE anion exchange chromatography. Intactness of functional binding domains was evaluated by measuring the retention of steroid- and DNA-binding capacity. Under non-denaturing conditions the intact AR is a highly asymmetrical molecule with a Stokes radius (RS) of 45A, a sedimentation coefficient of 4.3S and a relative molecular mass of 80,000 daltons. This form of AR has an intrinsic binding affinity for DNA and was eluted from DNA-cellulose with 9 mM MgCl2. Chymotrypsin produced a more globular polypeptide (RS: 31A; 3.1S; 41,000 daltons) with a decreased net negative charge. This fragment also displayed DNA-binding affinity but required a higher concentration of MgCl2 (14 mM) for DNA-cellulose elution, indicating an increased affinity for DNA. The observed reduction in molecular size upon chymotrypsin treatment was confirmed when analysed by SDS-polyacrylamide gel electrophoresis after covalently labelling of the AR with [3H]R1881. Rat prostate cytosol contains a protease which is very active in generating an AR polypeptide with an increased affinity for DNA, without changing the AR net negative charge (RS: 33A; 3.7S; 51,000 daltons). The specificity of this protease remained unknown since none of a large number of inhibitors was able to inactivate this enzyme. The fragment generated is different from that obtained with chymotrypsin since significant differences in size as well as in charge were measured. Trypsin treatment generated a much smaller polypeptide (RS: 25A; 2.9S; 30,000 daltons) which had lost its DNA-binding capacity, but not its steroid binding site. This form probably represents the so-called meroreceptor. When intact AR was treated sequentially with prostate cytosol and trypsin, a polypeptide fragment with identical properties was obtained, indicating the spatial separation of two of the proteolytic cleavage sites. These studies provide evidence for the distinct nature of the molecular domains for androgen and DNA interaction on the calf uterine AR.

Animals↗
PubMed1986

Differential effects of molybdate on the hydrodynamic and DNA-binding properties of the non-activated and activated forms of the androgen receptor in calf uterus.

Calf uterine cytosol contains an androgen receptor with a relative molecular mass of approx. 90,000. In this study we have analysed the structure and aggregation properties of the androgen receptor, using sucrose density gradient centrifugation on a vertical rotor (VTi65). In the presence of 10 mM NaCl the androgen receptor in whole cytosol sedimented at 8 S irrespective of the presence of molybdate. In 400 mM NaCl the receptor dissociated to a 4.3 S entity. In whole cytosol molybdate promoted a partial shift of the 4.3 S receptor into the aggregated 8 S state. The time of exposure of the receptor to molybdate and NaCl determined the proportion of receptor sedimentating at 8 S and 4.3 S. The DNA-binding form of the uterine androgen receptor when analysed under the conditions of the DNA-cellulose binding assay, sedimented at 6.5 S. Increasing concentrations of molybdate shifted its sedimentation coefficient gradually from 6.5 S to 4.5 S and in parallel reduced the DNA-binding capacity. Molybdate added to a partially purified, DNA-binding form of the androgen receptor did not promote receptor aggregation to faster sedimentating forms. This suggests that such preparations are devoid of an androgen receptor-aggregation factor. Indirect evidence for such a factor was obtained from reconstitution experiments with whole cytosol. Our results indicate that the DNA-binding form of the androgen receptor interacts with a cytosol factor to form the 8 S receptor complex. Molybdate has diverse effects: in the presence of the cytosol factor it stabilizes the 8S complex; in its absence molybdate prevents in a concentration-dependent way DNA-binding as well as reaggregation of the monomeric 4.3 S form.

Animals↗
PubMed1986

Androgen-dependent growth regulation of and release of specific protein(s) by the androgen receptor containing human prostate tumor cell line LNCaP.

Hormone sensitivity, as indicated by the presence of steroid hormone receptors and by the effect of androgens and anti-androgens on the release of proteins by cultured cells of the human prostate tumor cell line lymph node carcinoma of the prostate-fast growing colony (LNCaP-FGC), has been studied. The growth of the LNCaP-FGC cells were stimulated by androgens in a dose-dependent way. Under optimal conditions the synthetic nonmetabolizable androgen 17 beta-hydroxy-17 alpha-methyl-(3H)-estra-4,9,11-trien-3-one (R1881) (0.1 nM) stimulated cell growth by approximately 2.3 times. Increasing doses of R1881 (1-100 nM) partly decreased the stimulation of the cell growth. The anti-androgen cyproterone acetate exerted inhibitory effects on cell growth. The nuclear extract of the LNCaP-FGC cells contained 17,000 +/- 2,500 (mean +/- SD) KCl-extractable, nuclear androgen receptor sites/cell. Estrogen and progesterone receptors were not detectable in the nuclear extracts nor in cytosol, indicating that these receptors were absent. The release of proteins in the culture medium was studied using incorporation of the 35S-methionine, sodium dodecyl sulfate (SDS)-gel electrophoresis, and fluorography. Cells grown in media containing charcoal-stripped fetal calf serum released significantly lower amounts of a protein with an apparent molecular mass of 42,000 daltons. The release of this 42-kD protein could be restored in cells cultured in the presence of 5 alpha-dihydrotestosterone (0.1-1 microM) or R1881 (0.1-100 nM), whereas the addition of estrogens or corticosteroids had no effect. In the presence of anti-androgens, such as cyproterone acetate and 5,5-dimethyl-3-(4-nitro-3-(trifluoro-methyl)-phenyl)-2,4-imidasolidin edione, inhibitory effects on the release of the 42-kD protein were observed. The observed parallel between the effects of (anti)-androgens on the growth of the LNCaP prostate cells and the release of the 42-kD protein suggests that this protein is involved in the regulation of malignant prostate cell growth.

Androgens↗
PubMed1986

Characterization of androgen receptors after photoaffinity labelling with [3H]methyltrienolone (R1881).

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in rat prostate, in a human transplantable prostatic adenocarcinoma (PC-82) and in calf uterus. Androgen receptors preparations were partially purified either via differential chromatography on 2',5'-ADP-Sepharose (rat prostate), via anion exchange fast protein liquid chromatography (rat prostate and PC-82) or via DNA-cellulose chromatography (calf uterus). Purification factors obtained with the three different methods were: 245, 75 and 40 respectively. Photolabelling of receptor preparations was performed via irradiation with a high pressure mercury lamp either before or after partial purification. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of approx 95 kD. The covalent attachment of [3H]R1881 to the 95 kD protein could be completely suppressed by a 200-fold molar excess of dihydrotestosterone. In rat prostate cytosol an androgen receptor with a molecular mass of approx 50 kD could be photoaffinity labelled with R1881. A similar size was found for the androgen receptor in the human prostatic adenocarcinoma. Our results show that photoaffinity labelling of androgen receptors with [3H]R1881 as ligand can be applied for characterization of partial purified androgen receptor preparations.

Affinity Labels↗
PubMed1986

Characterization of the calf uterine androgen receptor and its activation to the deoxyribonucleic acid-binding state.

The androgen receptor (AR) from calf uterine cytosol has been studied in terms of steroid-binding affinity, hormone dissociation kinetics, and DNA-cellulose-binding capacity. The binding affinity for three androgens, analyzed under conditions where binding to progesterone receptor did not occur, decreased in the order: methyltrienolone greater than 5 alpha-dihydrotestosterone greater than testosterone. Activation of the receptor to the DNA-binding state involved the following changes of the receptor: decrease in dissociation rate for the steroid, disaggregation of the receptor, and increase in affinity for DNA. Dissociation studies with methyltrienolone and 5 alpha-dihydrotestosterone revealed that the AR can exist in two affinity states which differ 13- to 30-fold in their affinity for the steroid. Molybdate (10-20 mM) prevented the formation of the high affinity state. The high affinity state receptor was formed in the absence of molybdate or after ammonium sulfate precipitation (0-40% saturation) of the molybdate-stabilized low affinity state receptor. During formation of the high affinity state, the sedimentation coefficient of the receptor in low ionic strength buffer decreased from 8-9S to 4.5S, indicating receptor disaggregation. DNA-cellulose binding capacity increased from 3 to 65% upon formation of the high affinity state. The DNA-binding form could be eluted from DNA-cellulose at 0.14 M NaCl. After elution the DNA-binding form maintained its sedimentation coefficient of 4.5S and chromatographed as a protein with a Stokes radius of 44 A. From these results it can be concluded that the activated, DNA-binding form of the AR in calf uterus is a protein with a molecular mass of approximately 85,000, which acquires a higher affinity for the ligand upon its formation.

Ammonium Sulfate↗
PubMed1986

Search for estrogen receptors in human meningioma tissue sections with a monoclonal antibody against the human estrogen receptor.

Meningiomas are rich in progestin receptors, whereas estrogen receptors (ER) are virtually undetectable. The present experiments were performed to evaluate whether the absence of ER from the majority of human meningioma cytosols can be attributed to: occurrence of only a small number of ER-positive cells in an otherwise ER-negative tissue; resistance of nuclear receptors to extraction; or impairment of steroid binding. Twenty-one specimens were selected from our total series of 67 meningiomas. Based on cytosol assays, five of these meningiomas were considered to be ER positive (10-42 fmol/mg protein) and five had marginal ER concentrations (4-9 fmol/mg protein), whereas the remaining tissues were ER negative. For comparison, human breast cancer tissues were used. Tissues were sectioned at 6 micron and stained immunocytochemically using a monoclonal antibody against the human ER. The breast cancer specimens showed specific nuclear staining in part of the tumor cells. The sensitivity of the immunocytochemical assay was found to be sufficient to detect staining in breast cancer tissues containing as little as 17 fmol ER/mg cytosol protein. No specific staining was observed in meningioma tissues. It is concluded that the majority of meningiomas are truly devoid of ER and that the estrogen binding agent detected in low concentration in some meningioma cytosols is immunologically different from the human breast cancer ER. The presence of progestin receptors in meningioma apparently does not require the continuous presence of ER.

Antibodies, Monoclonal↗