Studies of pituitary lactogenic hormone. Physicochemical characterization on procine prolactin.

Porcine pituitary lactogenic hormone has been examined by exclusion chromatography, analytical ultracentrifugation, free-boundary electrophoresis, solubility behavior, circular dichroism, and fluorescence spectra. The results have been compared with previous studies of ovine pituitary lactogenic hormone. These two hormones were found to resemble one another very closely by all criteria studied. While the homology between these lactogenic hormones and growth hormones is strongly reflected in the sharing of certain physicochemical properties, a number of distinct structural differences between these two closely related evolutionary lines are now becoming apparent.     

THE MOLECULAR EVOLUTION OF PITUITARY HORMONES

1. The pituitary hormones can be divided into 4 families; within each the members are structurally related and have probably evolved from a common ancestor by a process of gene duplication and divergence. 2. Recent structural studies have revealed much about the evolution of proteins. The roles of point mutation, gene duplication and partial gene duplication in molecular evolution have been highlighted, and the nature of the evolutionary forces involved has been extensively debated. The information available about the evolution of proteins in general provides a background for consideration of pituitary hormone evolution. 3. The structure and function of the mammalian neurohypophysial hormones (oxytocin and vasopressin) has been studied in detail. Related (structurally similar) peptides have been found in the neurohypophyses of lower vertebrates and have been Characterized in many instances. Several schemes have been proposed for the evolution of these hormones. 4. The vasopressins of the pig and its relatives show a genetic polymorphism. The roles of neurohypophysial hormones in lower vertebrates are very varied and not fully understood. 5. The ACTHs and MSHs are members of a second family of pituitary hormones. They are polypeptides of moderate size. Studies on amino-acid sequences have been carried out for ACTHs and MSHs from several mammals. α-MSH is identical in all cases studied in detail, but β-MSH and ACTH vary to some extent. There is considerable sequence homology between the hormones in this family - indicating a common phylogenetic origin and several gene duplications. 6. Dogfish MSH is the only non-mammalian hormone of the ACTH-MSH family to have been studied in detail. Two MSHs have been isolated from this species; both resemble the a-MSH of mammals in amino-acid sequence. ACTH-like and MSH-like hormones exist in many other vertebrate groups, but have not been characterized fully. 7. Structure-function relationships have been widely studied in the ACTH-MSH family, and have some interesting evolutionary implications. Polymorphism of P-MSHs is found in some mammals. 8. A third family of protein hormones includes pituitary prolactin and growth hormone, and placental lactogen. These are proteins of moderate size which have been shown to be widely distributed among the vertebrates. Species specificity can be recognized with regard to biological, immunological and structural properties. 9. Amino-acid sequences have been determined for growth hormones and prolactins from several mammals. There is sequence homology between growth hormone and prolactin. Human placental lactogen closely resembles human growth hormone. A phylogenetic tree has been constructed for this protein family. Rates of evolution within the group are rather variable. 10. The fourth family of pituitary hormones (FSH, LH, TSH and some related placental hormones) are all glycoproteins and have a subunit structure. Extensive sequence studies have been carried out on the hormones from some mammals, and show that there is considerable homology between the various subunits. The α-subunits of human TSH, LH and HCG (and probably FSH) are very similar. The β-subunits are different, but homologous. Evolution of this family clearly took place by a series of gene duplications followed by gene divergence. Schemes whereby this could have occurred have been discussed. Related hormones occur in lower vertebrates, but have not been fully characterized. Some lower vertebrates may possess only one gonado-trophin. 11. The pituitary hormones provide an interesting range of evolutionary problems, and are useful models for the study of molecular evolution. The evolutionary processes involved in their diversification have been discussed, with particular reference to the co-evolution of hormones and their receptors. Neutral mutations and gene duplications may have played a role in providing co-existing variation of hormones and receptors. 12. A speculative model for the evolution of neurohypophysial hormones is proposed, as an example of how molecular evolution may have operated in this and other hormone groups. 13. Homologies have been proposed between the various families of pituitary hormones, and between pituitary proteins and other entero-secretory proteins. The pituitary protein hormones were probably elaborated from smaller molecules by a process of partial gene duplication.     

Equilibrium denaturation of human growth hormone and its cysteine-modified forms

The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was studied. Denaturation was measured by ultraviolet absorbance, intrinsic fluorescence, far ultraviolet circular dichroism, and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165 (large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism. The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state. The folding results with hGH and alkylated hGH substantiate this.     

Isolation and partial characterization of secreted mouse pituitary prolactin.

Prolactin secreted by mouse anterior pituitaries was purified by gel filtration on Sephadex G-100. Electrophoretic homogeneity of the purified hormone was demonstrated in several gel systems, and electrophoresis in the presence of sodium dodecyl sulfate indicated an apparent molecular weight of 21 000 +/- 2000. Mouse and ovine prolactin displayed parallel dose vs. response curves in radio-receptor binding studies, indicating that these two hormones compete for identical receptor sites on rabbit mammary membranes. Comparative peptide mapping studies carried out on tryptic digests of mouse and ovine prolactin suggested only partial homology between the two hormones. Internally labeled monomeric mouse prolactin was observed to undergo aggregation following storage at --20 degrees C for 2 months.     

Primary structure of equine pituitary prolactin

Equine prolactin was determined to be a single chain protein of 199 amino acid containing two tryptophan and six cysteine residues, as found in other mammalian prolactins. The primary sequence of equine prolactin was obtained by automated Edman analyses of S-carboxymethylated protein and proteolytic fragments of modified protein. Of the known prolactin sequences, equine prolactin shows closest homology with porcine (93%) and fin whale (87-91%) prolactins. Genetic mutations have produced changes in 17 of 199 residues of equine prolactin relative to its putative ancestral precursor. Since equine growth hormone has undergone alterations in 4 of 191 residues relative to this putative precursor protein, these results support the theory that prolactins are evolving at a faster rate than growth hormones. Consistent with the previously determined circular dichroic spectrum of equine prolactin, 60% of the protein is predicted to form alpha helices.     

A new microbioassay for the measurement of lactogenic hormones in human serum

The standard Nb2 assay for biologically active prolactin has been modified to allow a rapid convenient microbioassay without loss of specificity or accuracy. Lactogenic hormones specifically stimulate the replication of Nb2 node rat lymphoma cells in suspension culture and form the basis of a currently available bioassay to measure prolactin and growth hormone in human serum. A new microbioassay was developed using microtest plates enabling a large number of samples to be assayed simultaneously whilst maintaining the overall sensitivity of the bioassay for lactogenic hormones. Growth of the Nb2 node lymphoma cells, measured by a light scattering technique using optical density on a spectrophotometer, was shown to be closely correlated with the cell number determined on a Coulter counter. Addition of excess anti-human prolactin and anti-human growth hormone completely inhibited the growth stimulatory effects of both human prolactin and human growth hormone. This new microbioassay (BA) and conventional radioimmunoassay (RIA) were used to measure lactogenic hormones in 48 normal subjects. There was a close correlation between the results of both assays for each hormone studied in the control sera. The mean basal BA/RIA ratio was 1.5 (range 0.8-2.0) for prolactin, 0.7 (range 0-4.5) for growth hormone and 1.3 (range 0.5-1.9) for total lactogenic activity.     

Human growth hormone DNA sequence and mRNA structure: possible alternative splicing.

We have determined the complete sequence of the human growth hormone (hGH) gene and the position of the mature 5' end of the hGH mRNA within the sequence. Comparison of this sequence with that of a cloned hGH cDNA shows that the gene is interrupted by four intervening sequences. S1 mapping shows that one of these intervening sequences has two different 3' splice sites. These alternate splicing pathways generate hGH peptides of different sizes which are found in normal pituitaries. Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones.     

New mechanisms of hormone secretion: MDR-like gene products as extrusion pumps for hormones?

P-glycoprotein, the product of the multidrug resistance (MDR1) gene, is an ATP-driven transmembrane pump that increases the resistance of cells by actively exporting toxic chemicals. In addition to transporting anticancer drugs, P-glycoprotein has been reported to extrude a variety of lipophilic drugs, such as calcium channel blockers, phenothiazines, cyclosporines etc. Interestingly, recent experiments suggest that steroid hormones may be physiologic substrates for P-glycoprotein. In addition, there exists a family of transporter genes with high structural homology to P-glycoprotein, the so-called ABC (ATP-binding casette) family. Although the physiological ligands for most of these transporters are unknown, there is increasing evidence that peptides may be transported by some of these proteins. Thus, the a-factor, a farnesylated pheromone with 13 amino acids, is exported from yeast cells by the product of the STE6 gene, a transporter protein with high homology to P-glycoprotein. Recently, we have cloned a novel member of the ABC-transporter gene family from neuroblastoma x glioma hybrid (NG-108-15) cells. This putative transporter gene ("NG-TRA") is expressed in the adrenal gland, kidney and in the brain. High amounts of NG-TRA mRNA are found in a variety of human brain tumors. Whether NG-TRA and/or other MDR-related transporters are involved in the transport of steroids, peptide hormones or growth factors remains to be established. If so, the cellular export of hormones by active pumps may represent a new mechanism of hormone secretion.     

Growth factor-mediated regulation of aromatase activity in human skin fibroblasts.

We investigated the effects of various hormones and growth factors on aromatase activity in cultured human skin fibroblasts. Several potential trophic factors were tested for their ability to modify basal aromatase activity or the response to dibutyryladenosine 3',5'-cyclic monophosphate and dexamethasone because (i) no endogenous ligand has been identified that is responsible for stimulating aromatase activity in the periphery, and (ii) dexamethasone and cAMP analogs can increase this enzyme's activity in fibroblasts. The effect of insulin and insulin-like growth factors were examined in closer detail because of the clinical association between insulin and hyperandrogenism. Pituitary hormones and hypothalamic releasing factors, such as human ACTH (10 nM), beta-endorphin (10 nM), beta-lipotropin (10 nM), alpha-MSH (10 nM), gamma 3-MSH (10 nM), ovine luteinizing hormone (10 ng/ml), ovine follicle-stimulating hormone (10 ng/ml), ovine thyroid-stimulating hormone (10 ng/ml), rat growth hormone (10 ng/ml), rat prolactin (10 ng/ml), rat corticotropin-releasing factor (10 nM), luteinizing hormone-releasing factor (10 nM), thyrotropin-releasing factor (10 nM), human growth hormone-releasing factor (10 nM), and somatostatin (10 nM), have no significant effects on aromatase activity. Porcine inhibin A (10 ng/ml) and porcine activin AB (10 ng/ml), two ovarian hormones with structural transforming homology to transforming growth factor-beta, also have no effect on aromatase activity. Although basic fibroblast growth factor (1-100 ng/ml), acidic fibroblast growth factor (1 ng/ml), epidermal growth factor (1 ng/ml), platelet-derived growth factor (1 ng/ml), tumor necrosis factor (1 ng/ml), and transforming growth factor-beta 1 (1 ng/ml) have no effect on basal aromatase activity in human skin fibroblasts, all of these growth factors inhibited the ability of dibutyryladenosine 3',5'-cyclic monophosphate to stimulate aromatase activity. In contrast, both insulin (100 pg/ml-10 ng/ml) and insulin-like growth factor-1 (1-100 ng/ml) had no effect on cAMP-stimulated aromatase but potentiated the action of dexamethasone (100 nM). Thus, there is a clear distinction between the effects of dexamethasone and cAMP on peripheral aromatase. On the basis of the results presented here, it is interesting to speculate that the hyperandrogenism that is often associated with insulin resistance may be due to a combination of growth factor-mediated inhibition of aromatase activity and the failure of peripheral tissues to respond to insulin and metabolize androgens to estrogens.     

The genes for human gastrin and cholecystokinin are located on different chromosomes

Summary The polypeptide hormones gastrin and cholecystokinin are structurally related, having the identical pentapeptide GWMDF located at their C-terminus. The precursors to these two hormones also show amino acid homology, suggesting that they may have a common ancestral origin. Recombinant DNA clones corresponding to gene fragments encoding human gastrin and cholecystokinin were used to determine their respective chromosomal localization by analyzing human-rodent cell lines. We have assigned the cholecystokinin gene to human chromosome 3q12-3pter and the gastrin gene to chromosome 17q.     

Assay of lactogenic hormones using receptors isolated from rabbit liver.

Using 125-I-labelled ovine prolactin and receptors isolated from the livers of rabbits, a sensitive method has been developed suitable for the assay of ovine, bovine, porcine, human and rat prolactins. These hormones showed competitive displacement of 125-I-ovine prolactin which was in general agreement with their respective activities in the pigeon crop sac bioassay. Human and monkey growth hormones and human placental lactogen, which have marked prolactin-like actions on mammary tissue were also effective competitors. Non-primate growth hormones (ovine, bovine, equine and canine) which do not have prolactin-like activity gave little if any displacement as did human FSH, LH, TSH, ACTH and bovine insulin. Preparations of equine and canine prolactin of varying purity gave dose-response curves although their activity as competitors relative to ovine prolactin was poor and not related to their pigeon crop stimulating activity. This indicates species differences between prolactins in hormone-receptor interaction. Experiments with antiserum to human growth hormone have suggested an effective method of making the assay specific in species such as man in which prolactin is not the sole hormone with lactogenic activity.     

Nucleotide sequence of a cDNA for the common alpha subunit of the bovine pituitary glycoprotein hormones. Conservation of nucleotides in the 3'-untranslated region of bovine and human pre-alpha subunit mRNAs

A cDNA clone for the pre-alpha subunit of the pituitary glycoprotein hormones has been isolated from a bovine pituitary cDNA library through the use of a pool of synthetic oligodeoxynucleotide probes. This clone, designated pB alpha, contains a 564-base pair insert which includes a portion of the signal sequence, the entire coding sequence of the mature protein, and 224 base pairs of the 3'-untranslated sequence. As expected, the nucleotide and amino acid sequence of the mature bovine alpha subunit was homologous to the sequences reported for humans and rodents, with the most extensive homology occurring between bovine and rodents (85-90%). However, a comparison of the 3'-untranslated regions of pre-alpha subunit mRNA from three different mammalian species indicated that in bovine and rat, or in human and rat, these sequences have rapidly diverged, yielding respective homologies of 21 and 36%. In contrast, the sequence homology observed between the 3'-untranslated regions of bovine and human was 79%, which approaches the level of homology shared by their coding sequences. Thus, the conservation of the 3'-untranslated sequence in bovine and human pre-alpha subunit mRNA may be an indication that this region is functionally significant in these two species.     

Purification to apparent homogeneity of a thymocyte specific growth factor from calf thymus

A thymic peptide previously found to recruit thymocytes from G1 into S phase has been purified from a crude thymic extract by subsequent steps of gel exclusion chromatography and reverse phase high performance liquid chromatography (HPLC). The purified material, which appeared homogeneous on thin-layer chromatography and HPLC, stimulated the DNA synthesis of cultured guinea pig thymocytes in a nanomolar concentration range. The amino acid composition revealed a high content of acidic amino acids and no apparent homology to previously defined growth factors and thymus differentiation hormones.     

黄花倒水莲生长与内源激素水平动态变化关系研究北大核心CSCD

为探索黄花倒水莲春梢生理生化特性的差异以及不同内源激素的变化规律,该文对黄花倒水莲春梢的生长动态进行监测,采用间接酶联免疫吸附法(ELISA)测定脱落酸(ABA)、生长素(IAA)、赤霉素(GA)、乙烯(ETH)和玉米素核苷(ZR)五种内源激素含量的动态变化,并对两者间的相关性进行分析。结果表明:(1)黄花倒水莲春梢生长发育过程可分为快速增长期(0~12 d)、生长转折期(16~20 d)和缓慢增长期(24~32 d)三个阶段。(2)内源激素ABA、GA、ETH和ZR含量在缓慢增长期显著高于快速增长期和生长转折期,IAA含量各时期差异较小。(3)春梢长、底部叶长和叶宽在快速增长期与ABA、GA、ETH和ZR含量呈负相关,且与ZR含量具有一定显著性,与IAA含量呈正相关;生长转折期,各指标与GA、ETH和ZR含量呈正相关,与GA含量具有一定显著性,与ABA含量呈负相关;缓慢增长期,各指标与五种内源激素含量均呈正相关,与IAA和ZR含量具有一定显著性。该研究结果为生产上利用外源激素调控黄花倒水莲春梢抽出以及生长提供了理论基础。     

Hormone/growth factor interactions mediating epithelial/stromal communication in mammary gland development and carcinogenesis

Epithelial/mesenchymal interactions begin during embryonic development of the mammary gland and continue throughout mammary gland development into adult life. Stromal and epithelial growth factors that may mediate interactions between these compartments of the mammary gland are reviewed. Since mammogenic hormones are the primary regulators of mammary gland development, special consideration is given to hormonal regulation of growth factors in order to explore the integration of hormones and growth factors in the regulation of mammary gland growth and neoplasia. Examination of hormonal regulation of the fibroblast growth factor (FGF)-7/FGFR2-IIIb receptor system in the mammary gland reveals that mammogenic hormones differentially regulate the synthesis of stromal growth factors and their epithelial receptors. These effects serve to optimize the action of estrogen and progesterone on mammary gland development and illustrate that the ratio of these two hormones is critical in regulating this growth factor axis. The role of stromal/epithelial mitogenic microenvironments in modulating the genotype and phenotype of preneoplastic and neoplastic lesions by chemical carcinogens is discussed. Finally, changes in growth factor expression during mammary tumor progression are described to illustrate the relative roles that stromally-derived and epithelial-derived growth factors may play during progression to hormone independent tumor growth.     

Growth hormone-releasing factor (GRF) stimulation test in the diagnosis of pituitary adenomas

The studies aimed at evaluation of pituitary reserve of growth hormone following stimulation with GRF have been carried out in a group of 33 patients (11 women and 22 men, of age between 25 and 62 years) with pituitary tumors. The studied material included cases with pituitary adenoma characterized by excessive secretion of growth hormone (somatotropinoma), prolactin (prolactinoma) or alpha subunits of glycoprotein hormones (alphoma), and those with hormonally inactive adenoma. The GRF stimulation tests were carried out in hospitalized patients after overnight fast between 8.00 and 10.00 a.m. Blood samples for hormonal determinations were taken before the test, and after 15, 30, 60, 90 and 120 minutes following intravenous administration of 100 micrograms of GRF 1-29. Besides growth hormone, also the blood serum concentrations of other pituitary hormones were determined in the patients studied, both in the basal state and during the dynamic tests. In patients with acromegaly the results of the determinations of growth hormone following stimulation with GRF showed considerable individual variability. In 5 cases there was an increase in blood serum growth hormone concentration. No response to GRF was noted in the remaining 8 cases. In adenoma cases of prolactinoma type, growth hormone concentration began to rise already at the 15-th minute of the test in most cases. In three cases of prolactinoma associated with acromegaly no response to GRF was observed. The cases of alphoma-type adenoma were usually characterized by the secretion of pituitary hormones other than growth hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of growth and chitosan production by Rhizopus oryzae in whey medium by plant growth hormones

The effect of some plant growth hormones, viz., gibberellic acid, indole-3-acetic acid, indole-3-butyric acid, and kinetin on chitosan production by Rhizopus oryzae in deproteinized whey was studied. Hormones, at different concentrations, increase the mycelial growth by 19-32%. However, increase in chitosan content of the mycelia was relatively small (1.7-14.3%) over the control. Maximum enhancement was observed with gibberellic acid. Fifty percent more chitosan could be obtained from 1L of whey containing 0.1mg/L gibberellic acid. Hormones, at higher dose, instead of stimulation inhibited both growth and mycelial chitosan content. This study showed that hormones have no influence on degree of deacetylation of chitosan but increase the quality of the chitosan by increasing weight average molecular weight and decreasing polydispersity. All the hormones had been found to enhance chitin deacetylase activity of R. oryzae by 1.067-1.267-fold and may be one of the reasons for increased chitosan production.