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Biomedical subjects

D B Lubahn

Publications and source records attributed to D B Lubahn.

At least 109 records · Page 6Linked to original sources
PubMed1990

Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity.

Mutations in the androgen receptor (AR) are thought to cause complete androgen insensitivity (CAIS) in 46,XY human subjects who have a female phenotype despite normal adult male concentrations of plasma testosterone. Assays of AR binding in cultured skin fibroblasts from subjects with CAIS show either an apparent absence of AR (AR-) or normal levels of AR (AR+) binding. In several subjects with CAIS, AR-, no gross AR mutation was detected by Southern blot analyses of genomic DNA and normal sized 10 kilobase mRNA was present on Northern blots of poly(A+) RNA from cultured genital skin fibroblasts. We have used the polymerase chain reaction to amplify individual exons within the human AR gene of subjects with CAIS and have identified point mutations in three subjects. In one AR- subject (R774C), amino acid 774 was changed from arginine (CGC) to cysteine (TGC), in another AR- subject (R831Q), arginine (CGA) was changed to glutamine (CAA) at position 831, and in an AR+ subject (V866M) a methionine (ATG) was substituted for valine (GTG) at position 866. Transfection of wild type and mutant AR cDNA clones into COS cells results in detection of AR protein by immunoblotting. AR ligand binding activity is absent in cells transfected with AR mutants R774C and R831Q, but present with AR mutant V866M. Androgen binding in cells transfected with AR mutant V866M has a 6-fold lower apparent binding affinity than that of wild-type AR. Transcriptional activation of the MMTV-CAT reporter gene was androgen dependent and specific and nearly maximal at physiological concentrations (10(-10) M) of androgen when wild-type AR was transfected into cells, whereas neither AR mutants R774C nor R831Q were able to stimulate CAT activity even at 10(-8) M androgen. AR mutant V866M was able to stimulate CAT activity but the androgen dose dependency was shifted toward pharmacological concentrations of steroid that exceed in vivo levels. The molecular basis of CAIS in humans exhibits genetic heterogeneity. Our study shows that some cases of CAIS are explained by an inability to form a functional AR-steroid complex and hence, the AR is unable to activate transcription of genes essential for male sex differentiation during fetal development.

Amino Acid Sequence↗
PubMed1990

Expression of recombinant androgen receptor in cultured mammalian cells.

Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.

Amino Acid Sequence↗
PubMed1989

Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity.

Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. We have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46,XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.

Alleles↗
PubMed1989

Identification of androgen receptors in normal human osteoblast-like cells.

The sex steroids, androgens and estrogens, are major regulators of bone metabolism. However, whether these hormones act on bone cells through direct or indirect mechanisms has remained unclear. A nuclear binding assay recently used to demonstrate estrogen receptors in bone [Eriksen, E.F., Colvard, D.S., Berg, N.J., Graham, M.L., Mann, K.G., Spelsberg, T.C. & Riggs, B.L. (1988) Science 241, 84-86] was used to identify specific nuclear binding of a tritiated synthetic androgen, [3H]R1881 (methyltrienolone), in 21 of 25 (84%) human osteoblast-like cell strains and a concentration of bound steroid receptors of 821 +/- 140 (mean +/- SEM) molecules per cell nucleus. Binding was saturable and steroid-specific. Androgen receptor gene expression in osteoblasts was confirmed by RNA blot analysis. Relative concentrations of androgen and estrogen receptors were compared by measuring specific nuclear estrogen binding. Nuclear binding of [3H]estradiol was observed in 27 of 30 (90%) cell strains; the concentration of bound estradiol receptor was 1537 +/- 221 molecules per cell nucleus. The concentrations of nuclear binding sites were similar in males and females for both [3H]R1881 and [3H]estradiol. We conclude that both androgens and estrogens act directly on human bone cells through their respective receptor-mediated mechanisms.

Binding, Competitive↗
PubMed1989

Cortisol alters gene expression during involution of the rat ventral prostate.

The ability of high doses of cortisol to retard the involution process in the rat ventral prostate was related to alterations in the pattern of gene expression. Poly(A)+ RNA preparations from the prostates of noncastrated, castrated, and castrated rats injected daily for 7 days with cortisol were compared by Northern blot hybridizations for the relative expression of genes associated with cell differentiation and maintenance (the C1 prostatic steroid binding protein gene and alpha-tubulin), with cell death (TRPM-2, hsp 70, and c-fos), and with hormone regulation (the androgen and glucocorticoid receptors). As anticipated, the concentration of C1 mRNA in the prostate fell to less than 4% of that in the noncastrated controls within 4 days after castration and was nearly undetectable after 7 days. This decline was retarded by cortisol treatment of 7-day castrated animals which sustained the level of C1 transcripts at approximately 50% of control. While the pattern of expression of alpha-tubulin indicated some minor fluctuations, with the highest level occurring 7 days after castration, the prostates of the cortisol-treated group had essentially the same concentration of this mRNA as the noncastrates. Cortisol also modified the expression of genes associated with prostatic cell death. The large increase in prostatic TRPM-2 mRNA, seen 7 days after castration, was reduced by over 80% after treatment with the glucocorticoid. Although not as abundantly expressed as TRPM-2, the castration-induced levels of transcripts for both hsp 70 and the protooncogene c-fos were substantially reduced by cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Androgen receptor locus on the human X chromosome: regional localization to Xq11-12 and description of a DNA polymorphism.

The gene for the androgen receptor, mutations at which cause the X-linked androgen insensitivity syndrome, has been localized to the q11----q12 region of the human X chromosome by analysis, using a cloned cDNA for the androgen receptor, of somatic cell hybrid panels segregating portions of the X chromosome. A moderate-frequency HindIII RFLP has been found which should be useful in genetic linkage analysis of the various inherited forms of androgen insensitivity.

Animals↗
PubMed1988

Cloning of human androgen receptor complementary DNA and localization to the X chromosome.

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.

Amino Acid Sequence↗
PubMed1988

Deletion of the steroid-binding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: evidence for further genetic heterogeneity in this syndrome.

The cloning of a cDNA for the human androgen receptor gene has resulted in the availability of cDNA probes that span various parts of the gene, including the entire steroid-binding domain and part of the DNA-binding domain, as well as part of the 5' region of the gene. The radiolabeled probes were used to screen for androgen receptor mutations on Southern blots prepared by restriction endonuclease digestion of genomic DNA from human subjects with complete androgen insensitivity syndrome (AIS). In this investigation, we considered only patients presenting complete AIS and with the androgen receptor (-) form as the most probable subjects to show a gene deletion. One subject from each of six unrelated families with the receptor (-) form of complete AIS and 10 normal subjects (6 females and 4 males) were studied. In the 10 normal subjects and in 5 of the 6 patients, identical DNA restriction fragment patterns were observed with EcoRI and BamHI. In one affected individual, a partial deletion of the androgen receptor gene involving the steroid-binding domain was detected. Analysis of other members of this family confirmed the apparent gene deletion. Our data provide direct proof that complete AIS in some families can result from a deletion of the androgen receptor structural gene. However, other families do not demonstrate such a deletion, suggesting that point mutations (or small, undetectable deletions) may also result in the receptor (-) form of complete AIS, adding further to the genetic heterogeneity of this syndrome.

Blotting, Southern↗
PubMed1988

The production of antibodies against the conserved cysteine region of steroid receptors and their use in characterizing the avian progesterone receptor.

The most highly conserved feature of steroid receptor primary structure, the conserved cysteine region, corresponds to the DNA-binding domain of steroid receptors. This domain of chick progesterone receptor (PR) and other receptors was immunochemically characterized using site-directed antibodies. Eight peptides were synthesized corresponding to portion of the conserved cysteine region from PR, glucocorticoid, or estrogen receptor proteins. Polyclonal antibodies were obtained by immunizing rabbits with peptide conjugated to a protein carrier. The cross-reactivity of each antiserum was tested by Western blotting against chick and human PR, human glucocorticoid receptor, and human estrogen receptor. Of the several antisera positive by Western blots, only one cross-reacted with native chick PR. It was found that antibody bound to 4S transformed receptor, but not to 8S nontransformed receptor. At 50 mM KCl, antibody bound preferentially to transformed receptor form A, but at 400 mM KCl antibody bound equally well to either receptor form A or B. Binding of PR to DNA-cellulose was partially inhibited by the presence of cross-reacting peptide antibody. Thus, we have obtained at least one site-directed antibody against steroid receptors which is a useful structural probe to the DNA-binding functional domain of these gene regulatory proteins.

Amino Acid Sequence↗
PubMed1988

Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor.

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.

Amino Acid Sequence↗
PubMed1988

The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate.

Androgenic hormones mediate their effects on male sex differentiation and development through a high affinity receptor protein. We report here cloning of the complete coding sequence of the human androgen receptor (hAR). By sequence homology hAR is a member of the nuclear receptor family, with closest sequence identity to the progesterone, mineralocorticoid, and glucocorticoid receptors. Regions of highest homology include the DNA-binding domain and a small region within the hydrophobic ligand-binding domain. Comparison of the deduced 919 amino acid sequence of hAR (98,999 mol wt) to the 902 amino acid sequence of rat AR (98,227 mol wt) reveals identical sequences in the DNA- and hormone-binding domains, with an overall homology of 85%. In human prostate, the major androgen receptor mRNA species is 10 kilobases while a less abundant mRNA is approximately 7 kilobases. Rabbit polyclonal antibodies were raised against a synthetic peptide from the N-terminal region of hAR. Immunocytochemical analysis of human prostate tissue demonstrated that AR is localized predominantly in nuclei of glandular epithelial cells.

Amino Acid Sequence↗
PubMed1988

The rat androgen receptor: primary structure, autoregulation of its messenger ribonucleic acid, and immunocytochemical localization of the receptor protein.

A composite androgen receptor DNA sequence 4,181 base pairs in length was determined from three cDNA clones isolated from a rat epididymal bacteriophage lambda gt11 library. An open reading frame of 902 amino acids encodes a protein of 98,227 mol wt. Structural domains characteristic of the steroid receptor family include an amino-terminal region with five repeated amino acid motifs, a central DNA-binding domain homologous with other steroid receptors, and a carboxyl-terminal steroid-binding region. A receptor cDNA probe used in Northern blot analysis hybridized with a predominant 10-kilobase androgen receptor mRNA in male reproductive tissues of the rat. Autoregulation of androgen receptor mRNA was indicated in rat ventral prostate by an increase in the level of 10-kilobase mRNA after castration and suppression of receptor mRNA upon androgen restimulation. A 15 amino acid peptide with sequence derived from the deduced androgen receptor sequence was synthesized and used as immunogen in raising receptor antibodies in rabbits. Antisera reacted with high titer against the synthetic peptide by enzyme-linked immunosorbent assay and against the native [3H]dihydrotestosterone-labeled androgen receptor as evidenced by an increase in receptor sedimentation rate determined by sucrose gradient centrifugation. Immunocytochemical staining localized the androgen receptor to epithelial cell nuclei in rat ventral prostate.

Amino Acid Sequence↗
PubMed1988

The Malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms.

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to less than 6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism--Thr:Ala--at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148thr) are designated Malmö A, and negative reactors (148ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and "lyonization" is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over--including double crossing-over--appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies approximately 50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.

Antibodies, Monoclonal↗
PubMed1987

Purification and characterization of mouse uterine estrogen receptor under conditions of varying hormonal status.

Mouse uterine estrogen receptor (ER) was purified about 11,000-fold from normal mouse uteri by affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified ER demonstrated a major component of 65,000 mol wt with minor fragments of the 54,000- and 37,000-dalton species, as judged by affinity labeling with [3H]tamoxifen aziridine and immunodetection with an ER monoclonal antibody (H-222). The minor fragments were not detected with additional monoclonals (H-226 or D-547), which recognize different domains on the ER molecule. Two-dimensional gel electrophoresis revealed that the major component with a mol wt of 65,000 had a pI of about 6.5. The 54,000- and 37,000-dalton components had similar pI values. Saturation binding and Scatchard plot analysis of purified ER yielded one class of binding sites with an apparent dissociation constant of about 1.4 nM. Changes in the hormonal status resulted in changes in the size of the ER even in the presence of molybdate and leupeptin.

Animals↗
PubMed1987

Population genetics of coagulant factor IX: frequencies of two DNA polymorphisms in five ethnic groups.

Two frequently used restriction-enzyme polymorphisms (RFLPs) of coagulant F.IX, TaqI and XmnI, have been examined in five ethnic groups: white Americans, black Americans, East Indians, Chinese, and Malays. There is a distinct "cline" in the frequencies of both polymorphisms, from white Americans to Malays. The rarer type 2 alleles of both polymorphisms, in which middle recognition sites are present--and which in our sample reach their highest frequencies in white Americans--are marginally higher in four groups of Europeans previously reported by others. The frequencies of the rarer alleles are significantly higher in Europeans than in black Americans and East Indians, and these alleles are essentially absent in Chinese and Malays. The frequency of heterozygosity diminishes in the same order, being zero in Malays for both polymorphisms. The polymorphisms are in strong linkage disequilibrium, and in all groups the type 1 allele for TaqI is disproportionately accompanied by the type 1 allele for XmnI. The paucity of type 2 alleles and the low rate of heterozygosity in four non-European groups suggest that the polymorphisms will be of little diagnostic value south of Gibraltar and east of Suez. This prediction is confirmed by the observed haplotype frequencies in the black American and the Oriental groups.

Alleles↗
PubMed1985

Electrophoretic characterization of purified bovine, porcine, murine, rat, and human uterine estrogen receptors.

The calf uterine estrogen receptor (E2R) in the presence of sodium molybdate has been purified, 7,000-fold by a single passage over an estradiol affinity column. A dominant 70,000-dalton band and two minor bands at 50,000 and 30,000 daltons were observed by electrophoretic analysis. These bands had been eluted using estradiol, sodium sulfocyanate, CHAPS, and HEPES (pH 7.4) with insulin as a carrier protein. The identities of the protein bands were initially confirmed by their failure to bind the affinity column when saturated with estradiol. This single step purification procedure was reproducible and rapid, with yields of 10-20%, providing 25% purity. Diffusion blot analysis, with specific 35S- and 125I-labeled monoclonal antibodies to E2R, confirmed that the 70,000-dalton band represented the estrogen receptor. Specificity was demonstrated by inhibition of binding of purified E2R by both estradiol and diethylstilbestrol but not testosterone, progesterone, corticosterone, aldosterone, or hydrocortisone. The relative binding affinity of the purified receptor was: ethynyl estradiol greater than 17 beta estradiol greater than estriol greater than or equal to estrone greater than or equal to 17 alpha-estradiol greater than mestranol. Pig, human, mouse, and rat uterine estrogen receptors were similarly purified with the affinity column. As with the calf uterine preparations, a dominant 70,000-dalton band with minor bands at 50,000 and 30,000 daltons was identified by diffusion blot analysis in all the species examined.

Animals↗
PubMed1984

A rapid silver-stain procedure for use with routine electrophoresis of cerebrospinal fluid on agarose gels.

Oligoclonal banding patterns, when present in cerebrospinal fluid (CSF) electrophoresed on agarose gels, are important in the diagnosis of multiple sclerosis and other demyelinating diseases. Typically, an 80-fold concentration of 2.5 mL of CSF has been needed before the oligoclonal banding pattern could be detected with Coomassie Brilliant Blue stain. This excessive volume requirement often is a problem, particularly in children. We describe here an improved silver-stain procedure with which we routinely can detect the oligoclonal banding pattern in 1 to 3 microL of unconcentrated CSF. The procedure is inexpensive and takes less than 45 min. Our method is quick, safe, sensitive, and easily performed in the clinical laboratory.

Cerebrospinal Fluid Proteins↗