ABSTRACT
BACKGROUND: In assisted reproductive technology, human chorionic gonadotrophin (hCG) is administered subcutaneously for the induction of oocyte maturation and ovulation. Our efforts to develop orally bioavailable luteinizing hormone (LH) receptor agonists have led to the discovery of Org 43553, a low molecular weight (LMW) LH receptor (LH-R) agonist. METHODS: Org 43553 was tested in vitro and in vivo in pre-clinical pharmacological models to demonstrate efficacy and oral availability. RESULTS: Org 43553 is a potent stimulator of the human LH-R in vitro (EC(50) 3.7 nM). In primary mouse Leydig cells, Org 43553 stimulated testosterone production. Pharmacokinetic analyses showed high oral bioavailability in rats (79%) and dogs (44%) with a shorter half-life compared with hCG (3.4 versus 5.6 h in the rat). Ovulation induction by Org 43553 was demonstrated in immature mice as well as in cyclic rats after single-dose oral administration (50 mg/kg). The ovulated oocytes were of good quality as demonstrated by successful fertilization and implantation of normal embryos. In male rats, testosterone production was substantially induced after oral administration. CONCLUSIONS: Org 43553 is the first LMW LH-R mimetic with demonstrated in vivo efficacy upon oral administration and could therefore replace subcutaneously administered hCG. The elimination half-life of Org 43553 is substantially shorter than hCG, which could potentially represent a clinical benefit in reducing the risk of ovarian hyperstimulation syndrome (OHSS).
Subject(s)
Ovulation Induction , Ovulation/drug effects , Pyrimidines/pharmacology , Receptors, LH/metabolism , Thiophenes/pharmacology , Administration, Oral , Animals , Caco-2 Cells , Chorionic Gonadotropin/metabolism , Dogs , Female , Humans , Leydig Cells/metabolism , Male , Mice , Molecular Weight , Ovarian Hyperstimulation Syndrome , Pyrimidines/administration & dosage , Rats , Rats, Wistar , Thiophenes/administration & dosageABSTRACT
The expression of HLA-G by invading trophoblasts suggests a role for this molecule in embryo implantation. Putative targets for HLA-G are the uterine natural killer cells (uNK) that are abundantly present at the time of implantation. Since NK cells are potent producers of a variety of cytokines, interaction with HLA-G may result in the production of cytokines involved in trophoblast differentiation or tissue remodelling. In the present study we investigated the effect of membrane-bound HLA-G (mHLA-G) on the uterine mononuclear cell population (UMC) as a whole and on uNK cells in particular by measuring proliferation and cytokine production [interferon-gamma (IFN-gamma)/vascular endothelial growth factor (VEGF)/leukaemia inhibitory factor (LIF)/interleukin-3 (IL-3)]. Uterine cells were isolated from endometrium of non-pregnant women at the time that the endometrium is thought to be receptive to implantation, and then co-cultured with HLA-class I(-)/HLA-class II(+) 721.221 B-LCL cells transfected with mHLA-G. HLA-G suppressed the alloproliferative response of unfractionated UMC to 721.221 cells. Also, IFN-gamma and IL-3 production was strongly reduced. In contrast, purified uNK cells were stimulated by mHLA-G. Proliferation as well as IFN-gamma production was increased after co-culture with mHLA-G transfected 721.221 cells. HLA-G stimulated VEGF production by UMC as well as purified uNK cells. LIF-levels were below the detection level of our enzyme-linked immunosorbent assay. In conclusion, our data show that mHLA-G stimulates proliferation and cytokine production by NK cells, while down-modulating the response of unfractionated UMC.
Subject(s)
HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Interferon-gamma/biosynthesis , Killer Cells, Natural/metabolism , Uterus/metabolism , Cell Division/physiology , Embryo Implantation/physiology , Female , HLA-G Antigens , Humans , Pregnancy , Vascular Endothelial Growth Factor A/biosynthesisABSTRACT
Human leukocyte antigen (HLA)-G, which is mainly expressed at the maternal-fetal interface, may play a role in the immune tolerance of the semi-allogenic fetus by the mother. Functional studies have shown that HLA-G is indeed a potential modulator of different immune responses. Therefore, it is of interest to study the level of expression of soluble HLA-G in several biological fluids derived from women with and without fertility problems. In order to measure soluble HLA-G, a reliable and sensitive HLA-G specific sandwich ELISA is required. Here, we describe such an ELISA in which G233 is used as the coating antibody and 56B as the detecting antibody. In comparison with two other assays, this assay shows highest responses to recombinant HLA-G and native HLA-G in primary trophoblast culture supernatant and high responses to HLA-G in amniotic fluid. No HLA-G in follicular fluid or preimplantation embryo culture supernatant could be detected.
Subject(s)
Enzyme-Linked Immunosorbent Assay , HLA Antigens/analysis , Histocompatibility Antigens Class I/analysis , Amniotic Fluid/metabolism , Antibodies, Monoclonal , Female , Follicular Fluid/metabolism , HLA-G Antigens , HumansABSTRACT
Bone morphogenetic protein-4 (BMP-4) plays an important role in the onset of endochondral bone formation in humans, and a reduction in BMP-4 expression has been associated with a variety of bone diseases. Here we describe, by transient transfection assays in bone cells, that the human BMP-4 promoter recently characterized in our laboratory can be stimulated specifically by antiestrogens but not by estrogens or other steroid hormones. This activity is dependent on the presence of the estrogen receptor (ER)-alpha, although the promoter lacks a consensus estrogen-responsive element. No activity was observed in the presence of ERbeta, but synergy was observed when both ER subtypes were cotransfected. The observed stimulation of BMP-4 promoter activity by antiestrogens appeared bone cell specific and was reversed upon addition of estrogens. Since antiestrogens are known to be effective in hormone replacement therapies for postmenopausal women, this observation may help to develop new strategies for treatment and prevention of osteoporosis.
Subject(s)
Bone Morphogenetic Proteins/genetics , Estrogen Receptor Modulators/pharmacology , Gene Expression Regulation/drug effects , Osteoblasts/drug effects , Promoter Regions, Genetic/drug effects , Raloxifene Hydrochloride/pharmacology , Adenocarcinoma/pathology , Base Sequence , Bone Morphogenetic Protein 4 , Bone Neoplasms/pathology , Breast Neoplasms/pathology , Cells, Cultured , Dimerization , Drug Design , Endometrial Neoplasms/pathology , Estrogen Receptor alpha , Estrogen Receptor beta , Estrogens , Female , Humans , Molecular Sequence Data , Neoplasms, Hormone-Dependent/pathology , Organ Specificity , Osteoblasts/metabolism , Osteogenesis/genetics , Osteoporosis/prevention & control , Osteosarcoma/pathology , Postmenopause , Receptors, Estrogen/chemistry , Receptors, Estrogen/drug effects , Receptors, Estrogen/genetics , Receptors, Estrogen/physiology , Recombinant Fusion Proteins/physiology , Stimulation, Chemical , Transfection , Tumor Cells, CulturedABSTRACT
Ever since the discovery of estradiol and the elucidation of its chemical structure, there has been a great deal of interest in its mechanism of action and its potential therapeutic value. It is now well established that estrogens have many different functions in many different cell-types. With respect to the potential use of estrogens as therapeutics, there is an interest in controlling reproductive function, bone metabolism, cardiovascular disease, as well as in the prevention of hot flushes, mood changes and Alzheimer s disease. For over a decade, it was believed that estrogens signal through a a single estrogen receptor, now referred to as ERalpha, which belongs to a family of ligand-activated transcription factors. More recently, however, a second estrogen receptor ERbeta was identified. The current review describes similarities as well as differences between these two distinct estrogen receptors. Both ERalpha and ERbeta bind 17beta-estradiol with high affinity and they bind to classical estrogen response elements in a similar if not identical fashion. However, there are also major differences between ERalpha and ERbeta for instance with respect to their tissue distribution, the phenotype of the corresponding knock-out mice and their transcriptional activities. It is anticipated that a better understanding of these two receptors will eventually lead to more selective ways of modulating physiological processes which are influenced by estrogens. For this purpose, the development of ERalpha and ERbeta specific ligands, both agonists as well as antagonists, will be of great importance.
Subject(s)
Estrogens/physiology , Receptors, Estrogen/physiology , Animals , Estrogen Receptor alpha , Estrogen Receptor beta , Female , Humans , Male , Receptors, Estrogen/drug effects , Receptors, Estrogen/geneticsABSTRACT
We have studied the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and chimeric constructs with ER alpha and ER beta to examine the bioactivities of these receptors and their responses to estrogen and antiestrogen ligands. Transcriptional activity of ER beta is highly dependent on cell/promoter context and on the nature of the ligand. ER beta activated significant levels of transcription in response to estrogens in certain cell types, but showed only moderate activity compared with ER alpha in others. Antiestrogens such as tamoxifen and 2-phenylbenzofuran, which show some agonistic activity with ER alpha, exhibit no agonistic activity with ER beta. Alteration of the amino-terminal A/B receptor domain can result in a dramatic change in cell type- and ligand-specific transcriptional activity of ER beta. Upon replacing the A/B domain of ER beta with the A/B domain of ER alpha, this receptor chimera not only exhibits an improved transcriptional response to estrogens, but also is now able to activate transcription upon treatment with these antiestrogens. As antiestrogen agonism was lacking in ER beta and the ER beta/alpha chimera containing the amino-terminal A/B domain of ER beta fused to domains C through F of ER alpha, but was restored in an ER alpha/beta chimera containing the A/B domain of ER alpha, antiestrogen agonism was shown to depend on the A/B domain (activation function-1-containing region) of ER alpha. Together, these results indicate that the differences in the amino-terminal regions of ER alpha and ER beta contribute to the cell- and promoter-specific differences in transcriptional activity of these receptors, and their ability to respond to different ligands, thus providing a mechanism for differentially regulated transcription by these two ERs.
Subject(s)
Receptors, Estrogen/biosynthesis , Transcription, Genetic/physiology , Amino Acid Sequence , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , CHO Cells , Cells, Cultured , Chimera/genetics , Cricetinae , DNA/biosynthesis , DNA/genetics , Female , Humans , Immunoblotting , Molecular Sequence Data , Receptors, Estrogen/genetics , Transcription, Genetic/genetics , Transfection/genetics , Transfection/physiologyABSTRACT
The estrogen receptor (ER) is expressed in two forms, ERalpha and ERbeta. Here we show that ERalpha and ERbeta, expressed both in vitro and in vivo, form heterodimers which bind to DNA with an affinity (Kd of approximately 2 nM) similar to that of ERalpha and greater than that of ERbeta homodimers. Mutation analysis of the hormone binding domain of ERalpha suggests that the dimerization interface required to form heterodimers with ERbeta is very similar but not identical to that required for homodimer formation. The heterodimer, like the homodimers, are capable of binding the steroid receptor coactivator-1 when bound to DNA and stimulating transcription of a reporter gene in transfected cells. Given the relative expression of ERalpha and ERbeta in tissues and the difference in DNA binding activity between ERalpha/ERbeta heterodimers and ERbeta it seems likely that the heterodimer is functionally active in a subset of target cells.
Subject(s)
DNA/metabolism , Receptors, Estrogen/genetics , Animals , Binding Sites , COS Cells , Deoxyribonuclease BamHI/metabolism , Dimerization , Electrophoresis, Polyacrylamide Gel , Estrogens/metabolism , Genes, Reporter , Histone Acetyltransferases , Humans , Kinetics , Nuclear Receptor Coactivator 1 , Transcription Factors/metabolism , Transcription, Genetic , TransfectionABSTRACT
A novel estrogen receptor (hereinafter referred to as ER beta) was cloned using degenerate PCR primers. A comparison of the amino acid sequence of ER beta with the "classical' ER (ER alpha) shows a high degree of conservation of the DNA-binding domain (96%), and of the ligand-binding domain (58%). In contrast, the A/B domain, the hinge region and the F-domain are not conserved. Northern blot analysis revealed that ER beta is expressed in human thymus, spleen, ovary and testis. Transient transfections of an ER beta expression construct together with an ERE-based reporter construct in CHO cells clearly demonstrated transactivation of ER beta by 17 beta-estradiol. In addition, the ER alpha antagonist ICI-164384 is a potent antagonist for ER beta as well. Interestingly, the level of transactivation by 17 beta-estradiol is higher for ER alpha than for ER beta, which may reflect suboptimal conditions for ER beta at the level of the ligand, responsive element or cellular context.
Subject(s)
Receptors, Estrogen/metabolism , Amino Acid Sequence , Base Sequence , Cell Line , DNA, Complementary , Estradiol/analogs & derivatives , Estradiol/metabolism , Estrogen Antagonists/metabolism , Estrogen Receptor beta , Female , Gene Expression Regulation , Genes, Reporter , Humans , Male , Molecular Sequence Data , Ovary/metabolism , Polyunsaturated Alkamides , Receptors, Estrogen/genetics , Sequence Homology, Amino Acid , Testis/metabolism , Thymus Gland/metabolismABSTRACT
Expression of the platelet-derived growth factor alpha-receptor (PDGFalphaR) gene is tightly controlled in mammalian embryogenesis. A well established model system to study human embryogenesis is the embryonal carcinoma cell line Tera2. We have shown previously that retinoic acid-differentiated Tera2 cells express two PDGFalphaR transcripts of 6.4 kilobase pairs (kb) (encoding the full-length receptor) and 3.0 kb, respectively, whereas in contrast, undifferentiated Tera2 cells express PDFGalphaR transcripts of 1.5 kb and 5.0 kb. Here we show that this switch in PDGFalphaR expression pattern during differentiation of Tera2 cells results from alternative promoter use. In undifferentiated cells, a second promoter is used, which is located in intron 12 of the PDGFalphaR gene. Functional analysis shows that this promoter contains a consensus octamer motif, which can be bound by the POU domain transcription factor Oct-4. Oct-4 is expressed in undifferentiated Tera2 cells but not in retinoic acid-induced differentiated cells. Mutation of the octamer motif decreases promoter activity, while ectopic expression of Oct-4 in differentiated Tera2 cells specifically enhances the activity of this PDGFalphaR promoter. Therefore, we suggest that an important aspect in the maintenance of the undifferentiated state of human embryonal carcinoma cells results from Oct-4 expression, which thereupon activates this PDGFalphaR promoter.
Subject(s)
Carcinoma, Embryonal/metabolism , DNA-Binding Proteins/metabolism , Promoter Regions, Genetic , Receptors, Platelet-Derived Growth Factor/genetics , Transcription Factors/metabolism , Alternative Splicing , Animals , Base Sequence , Binding Sites , Carcinoma, Embryonal/pathology , Consensus Sequence , Gene Expression Regulation, Neoplastic , Humans , Molecular Sequence Data , Octamer Transcription Factor-3 , Receptor, Platelet-Derived Growth Factor alpha , Tumor Cells, CulturedABSTRACT
Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.
Subject(s)
Biomarkers, Tumor/analysis , Gene Expression , Germinoma/pathology , Receptors, Platelet-Derived Growth Factor/biosynthesis , Testicular Neoplasms/pathology , Testis/metabolism , Transcription, Genetic , Adult , Alkaline Phosphatase/analysis , Base Sequence , Carcinoma, Embryonal , Choriocarcinoma/metabolism , Choriocarcinoma/pathology , Clone Cells , DNA Primers , Germinoma/classification , Germinoma/metabolism , Humans , Male , Molecular Sequence Data , Polymerase Chain Reaction , Receptor, Platelet-Derived Growth Factor alpha , Receptors, Platelet-Derived Growth Factor/analysis , Seminiferous Tubules/cytology , Seminiferous Tubules/metabolism , Seminiferous Tubules/pathology , Seminoma/metabolism , Seminoma/pathology , Teratoma/metabolism , Teratoma/pathology , Testicular Neoplasms/classification , Testicular Neoplasms/metabolism , Testis/cytology , Testis/pathology , Tretinoin/pharmacology , Tumor Cells, CulturedABSTRACT
Expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) is strictly regulated during mammalian development and tumorigenesis. The molecular mechanisms involved in the specific regulation of PDGF alpha R expression are unknown, but transcriptional regulation of the PDGF alpha R gene is most likely to be involved. This study describes the molecular cloning of the non-coding exon 1 and approximately 2 kb of 5' flanking region of the human PDGF alpha R gene. This 5' flanking region is a functional promoter of the PDGF alpha R gene as concluded from its capacity to drive luciferase reporter gene expression in an orientation dependent way. Analysis of 5' promoter deletion mutants revealed that the region from -441 to +118, relative to the transcription initiation site, is sufficient to establish high level promoter activity. In addition, the morphogen retinoic acid, alone or in combination with dibutyryl cAMP, gives a 22-fold induction of PDGF alpha R gene promoter activity in human teratocarcinoma cells. This effect is mediated through specific transcription factor binding within the -52/+118 region of the PDGF alpha R gene.
Subject(s)
Promoter Regions, Genetic , Receptors, Platelet-Derived Growth Factor/genetics , Base Sequence , Bucladesine/pharmacology , Cloning, Molecular , Humans , Molecular Sequence Data , TATA Box , Theophylline/pharmacology , Tretinoin/pharmacologyABSTRACT
Two novel platelet-derived growth factor (PDGF) alpha-receptor transcripts of 1.5 kilobases and 5.0 kilobases are expressed in the human teratocarcinoma cell line Tera-2 only while the cells are in an undifferentiated state. After retinoic acid-induced differentiation, expression of these mRNAs is completely shut off and instead, the cells express a single 6.4-kilobase mRNA species which is also expressed in many other cell types. The 1.5-kilobase mRNA initiates within intron 12, contains the correctly spliced exons 13, 14, 15, and 16, and contains a cryptic exon, designated teratocarcinoma cryptic exon, at the 3' end. Teratocarcinoma cryptic exon contains a functional polyadenylation signal. Exons 13 to 16 correspond to the first tyrosine kinase domain and to part of the interkinase domain of the PDGF alpha-receptor. Recently, a splice variant lacking exon 14 was identified. These results show that a combination of alternative promoter usage and alternative splicing of the human PDGF alpha-receptor gene occur in a developmentally regulated fashion. In vitro translation of the 1.5-kilobase mRNA generates protein products which can be specifically immunoprecipitated with a PDGF alpha-receptor-specific antibody. The significance of the expression of this transcript for the growth factor-independent proliferation of undifferentiated Tera-2 cells is unclear. Expression of PDGF alpha-receptor transcripts containing the cryptic exon may be useful as a marker for undifferentiated stem cells in human teratocarcinomas.
Subject(s)
Gene Expression Regulation, Neoplastic , Receptors, Platelet-Derived Growth Factor/analysis , Teratocarcinoma/chemistry , Amino Acid Sequence , Base Sequence , Chromosome Mapping , DNA, Neoplasm/analysis , Exons/genetics , Humans , Male , Molecular Sequence Data , RNA, Messenger/analysis , RNA, Messenger/genetics , Receptors, Platelet-Derived Growth Factor/genetics , Teratocarcinoma/pathology , Tumor Cells, CulturedABSTRACT
Type 2 (non-insulin-dependent) diabetes is associated with the deposition of islet amyloid. The major formative peptide, islet amyloid polypeptide, has recently been characterised and an abnormality of the structure or expression of this gene is a possible candidate for the inherited component of Type 2 diabetes. A restriction fragment length polymorphism of the gene has been identified with Pvu II. To study the relationship between the islet amyloid polypeptide gene and Type 2 diabetes, two distinct genetic approaches have been undertaken. Firstly, non-linkage has been demonstrated in four pedigrees, with four normoglycaemic first degree relatives having an allele associated with diabetes in other family members, and one affected relative not having the putatively associated allele. The LOD score taking age-related penetrance into account was -1.68, making linkage unlikely (p = 0.02). Secondly, in a population-based restriction fragment length polymorphism survey, no linkage disequilibrium of the alleles was found between a population of unrelated Caucasian subjects with Type 2 diabetes and a normal population. A mutation in or near the islet amyloid polypeptide gene is thus unlikely to be a common cause of Type 2 diabetes.
Subject(s)
Amyloid/genetics , Diabetes Mellitus, Type 2/genetics , Genetic Linkage , Polymorphism, Restriction Fragment Length , Blood Glucose/analysis , DNA/blood , DNA/genetics , DNA/isolation & purification , Deoxyribonucleases, Type II Site-Specific , Diabetes Mellitus, Type 2/blood , Female , Glucose Tolerance Test , Glycated Hemoglobin/analysis , Haplotypes , Humans , Islet Amyloid Polypeptide , Lod Score , Male , Middle Aged , PedigreeABSTRACT
Islet amyloid polypeptide (IAPP) or amylin is a pancreatic islet hormone which was first found in amyloid in insulinomas and in pancreases of patients with type 2 diabetes. In rat a similar polypeptide occurs; however, pancreatic amyloid in this species has not been described. Here we report the structure of the rat and human IAPP gene. Both consist of three exons and two introns which are very similar. The upstream sequence of the rat IAPP gene contains a TATA-box, a CCAAT-sequence and a GT-element, whereas the upstream sequence of the human IAPP gene contains a TATA-box and a rat insulin enhancer-like sequence. This suggests that the rat and human IAPP gene may be controlled differently at the transcriptional level.
Subject(s)
Amyloid/genetics , Transcription, Genetic , Animals , Base Sequence , Blotting, Northern , Enhancer Elements, Genetic , Exons , Genes , Humans , Insulin/genetics , Introns , Islet Amyloid Polypeptide , Molecular Sequence Data , Promoter Regions, Genetic , Rats , TATA BoxABSTRACT
Aberrant expression of the islet amyloid polypeptide (IAPP) gene might be involved in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM). Here, we report that IAPP promoter-luciferase constructs revealed tissue-specific activity. This activity was not mediated by cAMP. Sequential 5' deletions of the IAPP promoter caused a progressive derepression of the IAPP gene promoter in IAPP-producing cells. Comparison of the nucleotide sequence of the IAPP promoter with that of the insulin promoter (both active in pancreatic beta-cells) reveals two sequence elements of putative importance: an insulin enhancer-like sequence and an element which corresponds to a protected domain in rat insulin I gene promoter footprint experiments.
Subject(s)
Amyloid/genetics , Gene Expression Regulation , Islets of Langerhans/metabolism , Luciferases/genetics , Promoter Regions, Genetic , Base Sequence , Bucladesine/pharmacology , Chromosome Deletion , Colforsin/pharmacology , Gene Expression Regulation/drug effects , Insulin/genetics , Islet Amyloid Polypeptide , Islets of Langerhans/drug effects , Molecular Sequence Data , Promoter Regions, Genetic/drug effects , TATA Box , Tumor Cells, CulturedABSTRACT
Islet amyloid polypeptide (IAPP) is the 37-amino acid peptide subunit of amyloid found in pancreatic islets of type 2 diabetic patients and in insulinomas. Recently, we isolated the human gene encoding IAPP [(1988) FEBS Lett. 239, 227-232]. We now report the nucleotide sequences of a human insulinoma cDNA encoding a complete IAPP precursor, and of the corresponding parts of the IAPP gene. Two exons, which are approx. 5 kb apart in the human genome, encode the 89-amino acid pre-pro-IAPP. At least one additional exon is present further upstream in the IAPP gene. A putative signal sequence at the amino-terminus of the precursor suggests that IAPP is a secreted protein.
Subject(s)
Amyloid/genetics , Exons , Islets of Langerhans/analysis , Protein Precursors/genetics , Amino Acid Sequence , Base Sequence , Calcitonin/genetics , Calcitonin Gene-Related Peptide , Codon , DNA/genetics , DNA Probes , Humans , Insulinoma/analysis , Islet Amyloid Polypeptide , Molecular Sequence Data , Neuropeptides/genetics , Nucleic Acid Hybridization , Pancreatic Neoplasms/analysis , Protein Sorting Signals/genetics , Sequence Homology, Nucleic AcidABSTRACT
Islet or insulinoma amyloid polypeptide (IAPP) is a 37 amino acid polypeptide isolated from pancreatic amyloid. Here, we describe the isolation and partial characterization of the human gene encoding IAPP. The DNA sequence predicts that IAPP is excised from a larger precursor protein and that its carboxy-terminus is probably amidated. The predicted normally occurring IAPP is identical to the reported polypeptides isolated from pancreatic amyloid, except for the amidated carboxy-terminus. IAPP specific polyadenylated RNAs of 1.6 kb and 2.1 kb are present in human insulinoma RNA. The human IAPP gene is located on chromosome 12.
Subject(s)
Amyloid/genetics , Chromosomes, Human, Pair 12 , Genes , Amino Acid Sequence , Base Sequence , Blotting, Northern , Blotting, Southern , Chromosome Mapping , Humans , Islet Amyloid Polypeptide , Molecular Sequence Data , Nucleic Acid HybridizationABSTRACT
The insulin-like growth factors I and II (IGF-I and -II) are polypeptides which play an important role in growth and development of the organism. In the present report we describe the detection of human IGF-I RNAs (both type Ia and type Ib) and IGF-II RNAs in benign (leiomyoma) and malignant (leiomyosarcoma) tumours from smooth muscle origin, using Northern blot hybridization analysis. In normal smooth muscle tissue of the uterus we found low levels of IGF-I RNAs only. In the tumours the same IGF-I RNA species were detected as in adult non-tumour tissues (uterus, liver). For transcription of the IGF-II gene in these tumours, two promoters are used which are expressed in fetal liver, but not in adult liver. The presence of IGF-I and IGF-II RNAs was also established by nucleotide sequence analysis of recombinant DNA clones isolated from cDNA libraries derived from two leiomyosarcomas. The nucleotide sequences of these cDNA clones, together covering the entire coding regions of IGF-Ia and IGF-II var RNA, predict that IGFs encoded by the tumour RNAs do not differ in amino acid sequence from the corresponding polypeptides isolated from serum. In those tissues containing IGF-I RNAs, IGF-I immunoreactivity was also demonstrated.
Subject(s)
Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor I/genetics , Leiomyoma/genetics , Leiomyosarcoma/genetics , Somatomedins/genetics , Exons , Female , Gene Expression Regulation , Humans , Muscle, Smooth/metabolism , Promoter Regions, Genetic , RNA, Neoplasm/genetics , Uterine Neoplasms/geneticsABSTRACT
The schistosome parasite, Trichobilharzia ocellata, nearly completely inhibits the reproductive activity of its intermediate host, Lymnaea stagnalis. The synthetic activity of albumen glands of infected snails at day 35 postinfection (p.i.) is only 1% of the control value. The parasite acts by humoral means. We tested the hypothesis that (a) specific humoral agent(s) is (are) involved and refer to this (these) agent(s) as schistosomin. The presence of schistosomin in the hemolymph of infected snails was investigated by using galactogen synthesis in albumen glands as an in vitro bioassay. The synthetic activity of albumen glands of noninfected snails decreased by about 50% during a 1-h incubation in the hemolymph of infected snails. This inhibition is attributed to schistosomin. Based on these results, with the present bioassay schistosomin appears in the hemolymph between days 28-36 p.i. onwards. Schistosomin is heat-stable (100 degrees C) and pronase-sensitive, and therefore it might have a peptide nature. Schistosomin suppresses the stimulating action of the female, gonadotrophic dorsal body hormone at relatively low doses, which suggests that it may compete with this hormone for the same receptors. The development of two other bioassays for schistosomin in our laboratory is discussed.