Identification and characterization of a novel heparan sulfate-binding domain in Activin A longest variants and implications for function.

Activins regulate numerous processes including inflammation and are synthesized as precursors consisting of a long N-terminal pro-region and a mature protein. Genomic human databases currently list three activin A (Act A) variants termed X1, X2 and X3. The X3 variant is the shortest, lacks N-terminal segments present in X1 and X2, and has been the focus of most past literature. Here, we asked whether these variants are expressed by human cells and tissues and what structural features are contained within their pro-regions. Human monocytic-like cells THP1 and U937 expressed X1 and X2 variants after exposure to phorbol ester or granulocyte-macrophage colony-stimulating factor, while X2 transcripts were present in placenta. Expression vectors encoding full length X2 or X3 variants resulted in production and secretion of biologically active Act A from cultured cells. Previous studies reported a putative HS-binding domain (HBD) in the X3 pro-region. Here, we identified a novel HBD with consensus HS-binding motifs near the N-terminal end of X1 and X2 pro-regions. Peptides encompassing this new domain interacted with substrate-bound HS with nanomolar affinity, while peptides from putative X3 HBD did not. In good agreement, full length X2 pro-region interacted with heparin-agarose, while the X3 pro-region did not. In sum, our study reveals that Act A variants are expressed by inflammatory cells and placenta and yield biological activity. The high affinity HBD in X1 and X2 pro-region and its absence in X3 could greatly influence overall Act A distribution, availability and activity in physiological and pathological circumstances.

Polymorphisms of four candidate genes and their correlations with growth traits in blue fox (Alopex lagopus).

To improve the accuracy and genetic progress of blue fox breeding, the relationships between genetic polymorphisms and growth and reproductive traits of the blue fox were investigated. MC4R, MC3R, INHA and INHBA were selected as candidate genes for molecular evolution and statistical analyses. Single-factor variance analyses showed that the MC4R (g.267C > T, g.423C > T, and g.731C > A) and MC3R (g.677C > T) genotypes had significant impacts on body weight, chest circumference, abdominal perimeter and body mass index (BMI) (P < 0.05) in blue fox. The MC4R and MC3R combined genotypes had significant effects on the body weight and abdominal circumference. The different genotypes of INHA g.75G > A had significant effects on female fecundity, whereas the different genotypes of INHBA g.404G > T and g.467G > T and the INHA and INHBA combined genotypes had significant effects on male fecundity. The proteins encoded by the open reading frames (ORFs) of different polymorphic loci were predicted and analysed. The aims of this study were to identify genetic markers related to growth and reproduction in the blue fox and to provide an efficient, economical and accurate theoretical approach for auxiliary fox breeding.

Role of FSH glycan structure in the regulation of Sertoli cell inhibin production.

Variations in follicle-stimulating hormone (FSH) carbohydrate composition and structure are associated with important structural and functional changes in Sertoli cells (SCs) during sexual maturation. The aim of the present study was to investigate the impact of FSH oligosaccharide structure and its interaction with gonadal factors on the regulation of monomeric and dimeric inhibin production at different maturation stages of the SC. Recombinant human FSH (rhFSH) glycosylation variants were isolated according to their sialylation degree (AC and BA) and complexity of oligosaccharides (CO and HY). Native rhFSH stimulated inhibin α-subunit (Pro-αC) but did not show any effect on inhibin B (INHB) production in immature SCs isolated from 8-day-old rats. Activin A stimulated INHB and had a synergistic effect on FSH to stimulate Pro-αC. The less acidic/sialylated rhFSH charge analogues, BA, were the only charge analogue mix that stimulated INHB as well as the most potent stimulus for Pro-αC production. Native rhFSH stimulated both Pro-αC and INHB in SCs at a more advanced maturation stage, isolated from 20-day-old rats. In these cells, all rhFSH glycosylation variants increased INHB and Pro-αC production, even in the presence of growth factors. The BA preparation exerted a more marked stimulatory effect on INHB and Pro-αC than the AC. Glycoforms bearing high mannose and hybrid-type oligosaccharides, HY, stimulated INHB and Pro-αC more effectively than those bearing complex oligosaccharides, CO, even in the presence of gonadal growth factors. These findings demonstrate the modulatory effect of FSH oligosaccharide structure on the regulation of inhibin production in the male gonad.

Biological activity and in vivo half-life of pro-activin A in male rats.

Mature TGF-ß proteins are used in vivo to promote bone growth, combat obesity, reverse fibrosis and pulmonary arterial hypertension, and as potential rejuvenation factors. However, the serum half-life of this family of growth factors is short (∼5 min), limiting their therapeutic potential. Because TGF-ß proteins are normally secreted from cells with their prodomains attached, we considered whether these molecules could extend the in vivo half-life and activity of their respective growth factors. Using activin A as a model ligand, we initially modified the cleavage site between the pro- and mature domains to ensure complete processing of the activin A precursor. Co-immunoprecipitation studies confirmed mature activin A is secreted from cells in a non-covalent complex with its prodomain, however, the affinity of this interaction is not sufficient to suppress activin A in vitro biological activity. The plasma clearance profiles of purified pro- and mature activin A were determined over a 4 h period in adult male rats. Both activin forms demonstrated a two-phase decay, with the half-life of pro-activin A (t1/2 fast = 12.5 min, slow = 31.0 min) being greater than that of mature activin A (t1/2 fast = 5.5 min, slow = 20.3 min). Both pro- and mature activin A induced significant increases in serum follicle stimulating hormone levels after 4 h, but no differences were observed in the relative in vivo bioactivities of the two activin isoforms. Increased serum half-life of activin A in the presence of its prodomain identifies a new means to increase the therapeutic effectiveness of TGF-ß proteins.

Dynamics of bivalent chromatin domains upon drug induced reactivation and resilencing in cancer cells.

Epigenetic deregulation revealed by altered profiles of DNA methylation and histone modifications is a frequent event in cancer cells and results in abnormal patterns of gene expression. Cancer silenced genes constitute prime therapeutic targets and considerable progress has been made in the epigenetic characterization of the chromatin scenarios associated with their inactivation and drug induced reactivation. Despite these advances, the mechanisms involved in the maintenance or resetting of epigenetic states in both physiological and pharmacological situations are poorly known. To get insights into the dynamics of chromatin regulation upon drug-induced reactivation, we have investigated the epigenetic profiles of two chromosomal regions undergoing long range epigenetic silencing in colon cancer cells in time-course settings after exposure of cells to chromatin reactivating agents. The DNA methylation states and the balance between histone H3K4 methylation and H3K27 methylation marks clearly define groups of genes with alternative responses to therapy. We show that the expected epigenetic remodeling induced by the reactivating drugs, just achieves a transient disruption of the bivalent states, which overcome the treatment and restore the transcriptional silencing approximately four weeks after drug exposure. The interplay between DNA methylation and bivalent histone marks appears to configure a plastic but stable chromatin scenario that is fully restored in silenced genes after drug withdrawal. These data suggest that improvement of epigenetic therapies may be achieved by designing strategies with long lasting effects.

N-linked oligosaccharides direct the differential assembly and secretion of inhibin alpha- and betaA-subunit dimers.

The biosynthetic pathway governing inhibin heterodimer (alpha/beta) and activin homodimer (beta/beta) assembly and secretion from ovarian granulosa cells is not fully understood. Here, we examined the role of inhibin subunit glycosylation in the assembly and secretion of mature inhibin A and activin A. Inhibition of subunit glycosylation by tunicamycin treatment of alpha- and beta(A)-expressing CHO cell lines reduced inhibin but not activin secretion. Dimeric inhibin A is preferentially secreted from parental isogenic wild-type (wt) cell lines (alpha(wt)beta(wt)). Mutation of a single glycosylation site at asparagine 268 (alpha(Delta268)beta(wt)) reduces inhibin secretion by 78% and permits beta/beta assembly and secretion. Conversely, gain of a glycosylation (GOG) site in the analogous region of the beta(A)-subunit (alpha(wt)beta(GOG327)) enhances inhibin A secretion. The present study demonstrates that N-linked glycan sites direct heterodimer vs. homodimer assembly, and prevention of glycosylation abrogates inhibin secretion. These data support a definitive role for site-specific N-glycosylation in governing inhibin/activin dimer assembly and secretion.

Activin-A binds follistatin and type II receptors through overlapping binding sites: generation of mutants with isolated binding activities.

Follistatin is a potent extracellular antagonist of members of the TGFbeta superfamily that use activin type II receptors (ActRII/IIB) as part of their signaling complex. A recent crystallographic study indicates that follistatin contacts activin-A residues at both the type I (ALK4) and type II receptor binding interfaces. However, the relative contribution of these two sites on human activin-A to follistatin binding has not been determined. Residues at these sites were mutated to alanine and mutants were screened for their ability to bind follistatin and ActRII and induce FSH secretion from a gonadotrope cell line. Despite extensive mutagenesis across the type I receptor interface, activin-A affinity for follistatin was not significantly diminished. In contrast, mutagenesis of residues at the type II binding interface had pronounced effects on activin's interaction with follistatin. In particular, residues Leu92, Tyr94, Ile100, and Lys102 were critical for high-affinity follistatin binding. Interestingly, mutation of another primary determinant of ActRII/IIB binding, Ser90, did not affect follistatin affinity, suggesting that the interaction surfaces for type II receptors and follistatin were overlapping but not identical. In support, mutation of Asp95, on the opposite edge of the common ActRII/follistatin interface, was disruptive for follistatin binding without affecting ActRII/IIB interactions. Activin-S90A was able to compete with wild-type activin for follistatin binding, whereas activin-D95A, due to its 8-fold lower affinity for follistatin, is a potent activin agonist. These reagents could be used to modulate follistatin antagonism of activin and related ligands in processes such as cancer, wound healing, and reproduction.

The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding.

TGF-beta ligands stimulate diverse cellular differentiation and growth responses by signaling through type I and II receptors. Ligand antagonists, such as follistatin, block signaling and are essential regulators of physiological responses. Here we report the structure of activin A, a TGF-beta ligand, bound to the high-affinity antagonist follistatin. Two follistatin molecules encircle activin, neutralizing the ligand by burying one-third of its residues and its receptor binding sites. Previous studies have suggested that type I receptor binding would not be blocked by follistatin, but the crystal structure reveals that the follistatin N-terminal domain has an unexpected fold that mimics a universal type I receptor motif and occupies this receptor binding site. The formation of follistatin:BMP:type I receptor complexes can be explained by the stoichiometric and geometric arrangement of the activin:follistatin complex. The mode of ligand binding by follistatin has important implications for its ability to neutralize homo- and heterodimeric ligands of this growth factor family.

Structural basis for a functional antagonist in the transforming growth factor beta superfamily.

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.

Adenovirus-mediated overexpression of the activin betaC subunit accelerates liver regeneration in partially hepatectomized rats.

BACKGROUND/AIMS: The expression level of the activin betaC subunit is high in normal liver and reduces after partial hepatectomy, but its function is controversial. METHODS: To determine the role of the betaC subunit during liver regeneration, we overexpressed the betaC subunit gene in the liver by infusing adenovirus vector encoding the flag-tagged betaC subunit into the portal vein. Adenovirus vector encoding the beta-galactosidase was also infused as a control. Seventy percent hepatectomy was performed 4 days after the infection. RESULTS: Approximately 20% of hepatocytes expressed the flag-tagged betaC subunit at the time of hepatectomy and approximately 50% of hepatocytes expressed the betaC subunit 3 days after hepatectomy. In betaC-infected liver, bromodeoxyuridine labeling was significantly greater at 24 and 48 h after partial hepatectomy compared with the control liver. Consistent with this observation, the liver regeneration rate was significantly greater in betaC-transfected liver at 72 and 96 h after hepatectomy. Many of the bromodeoxyuridine-positive nuclei were observed in or by the betaC-transfected hepatocytes. CONCLUSIONS: These results indicate that liver regeneration is accelerated in betaC-overexpressing liver. The betaC subunit may function to promote replication of hepatocytes during liver regeneration.

A molecular recognition paradigm: promiscuity associated with the ligand-receptor interactions of the activin members of the TGF-beta superfamily.

The structure-function properties of the pleiotropic activins and their relationship to other members of the transforming growth factor-beta superfamily of proteins are described. In order to highlight the molecular promiscuity of these growth factors, emphasis has been placed on molecular features associated with the recognition by activin A and the bone morphogenic proteins of the corresponding extracellular domains of the ActRI and ActRII receptors. The available evidence suggests that the homodimeric activin A in its various functional roles has the propensity to fulfill key tasks in the regulation of mammalian cell behaviour, through coordination of numerous transcriptional and translational processes. Because of these profound effects, under physiologically normal conditions, activin A levels are closely controlled by a variety of binding partners, such as follistatin-288 and follistatin-315, alpha(2)-macroglobulin and other proteins. Moreover, the subunits of other members of the activin subfamily, such as activin B or activin C, are able to form heterodimers with the activin A subunit, thus providing a further avenue to positively or negatively control the physiological concentrations of activin A that are available for interaction with specific receptors and induction of cell signaling events. Based on data from X-ray crystallographic studies and homology modeling experiments, the molecular architecture of the ternary receptor-activin ligand complexes has been dissected, permitting rationalization in structural terms of the pattern of interactions that are the hallmark of this protein family.

Notch signaling modulates the nuclear localization of carboxy-terminal-phosphorylated smad2 and controls the competence of ectodermal cells for activin A.

Loss of mesodermal competence (LMC) during Xenopus development is a well known but little understood phenomenon that prospective ectodermal cells (animal caps) lose their competence for inductive signals, such as activin A, to induce mesodermal genes and tissues after the start of gastrulation. Notch signaling can delay the onset of LMC for activin A in animal caps [Coffman, C.R., Skoglund, P., Harris, W.A., Kintner, C.R., 1993. Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell 73, 659-671], although the mechanism by which this modulation occurs remains unknown. Here, we show that Notch signaling also delays the onset of LMC in whole embryos, as it did in animal caps. To better understand this effect and the mechanism of LMC itself, we investigated at which step of activin signal transduction pathway the Notch signaling act to affect the timing of the LMC. In our system, ALK4 (activin type I receptor) maintained the ability to phosphorylate the C-terminal region of smad2 upon activin A stimulus after the onset of LMC in both control- and Notch-activated animal caps. However, C-terminal-phosphorylated smad2 could bind to smad4 and accumulate in the nucleus only in Notch-activated animal caps. We conclude that LMC was induced because C-terminal-phosphorylated smad2 lost its ability to bind to smad4, and consequently could not accumulate in the nucleus. Notch signal activation restored the ability of C-terminal-phosphorylated smad2 to bind to smad4, resulting in a delay in the onset of LMC.

Expression of the inhibin/activin subunits alpha (alpha), beta-A (betaA) and beta-B (betaB) in benign human endometrial polyps and tamoxifen-associated polyps.

BACKGROUND: Inhibins (INH) are dimeric glycoproteins, composed of an alpha subunit (INH-alpha) and one of two possible beta subunits (INH-betaA or INH-betaB). They have substantial roles in human reproduction and in endocrine-responsive tumours. Therefore, the aims of this study were to determine the frequency and tissue distribution of INH-alpha, INH-betaA and INH-betaB in normal human endometrium and glandular-cystic endometrial polyps, and polyps caused by tamoxifen use. MATERIALS AND METHODS: Tissue samples were obtained from women in the proliferative, early secretory and late secretory phase as well as glandular-cystic polyps and endometrial polyps associated with tamoxifen use (n = 5 each). Immunohistochemistry with specific monoclonal antibodies, a semi-quantitative analysis and statistical evaluation was performed. RESULTS: INH-alpha, INH-betaA and INH-betaB were primarily observed in glandular and luminal epithelial cells, with a variant staining intensity in stromal cells. INH-alpha in glands was significantly higher during the early secretory phase (p < 0.05) and the late secretory phase (p < 0.01) than in the proliferative phase with a significant difference between the early secretory and the late secretory phases (p < 0.01). INH-betaA expression was significantly higher during the late secretory than the proliferative phase (p < 0.05) and the late secretory than the early secretory phase (p < 0.05), with no significant differences for INH-betaB. Glandular-cystic polyps showed significantly lower expression of INH-alpha and INH-betaA than the late secretory endometria (p < 0.05 and p < 0.01 respectively). Additionally, tamoxifen-associated polyps also demonstrated a significantly lower expression of INH-alpha and INH-betaA than late secretory endometria (p < 0.01 and p < 0.01 respectively). No statistical differences were observed between tamoxifen-associated and glandular-cystic polyps. DISCUSSION: INH-alpha, INH-betaA and INH-betaB were expressed in normal endometrium and endometrial polyps. A cyclical expression of INH-alpha and INH-betaA in normal glands may reflect a functional and hormone-dependent role in human endometrium. Significant differences in staining reaction between the late secretory endometria and polyps suggest that this tissue remains in the proliferating state rather than the secretory state. Therefore, endometrial polyps may be tumours of dysregulation with mainly proliferating characteristics, being unable to synchronise with normal endometrium.

Beta A versus beta B: is it merely a matter of expression?

Activins are members of the transforming growth factor (TGF) beta (beta) superfamily of proteins that function in a wide array of physiological processes. Like other TGFbeta ligands, activins are biologically active as dimers. An activin molecule is comprised of two beta-subunits, of which four isoforms have been identified: betaA, betaB, betaC, and betaE. The most widely studied activins to date are activin A (betaA/betaA), activin B (betaB/betaB), and activin AB (betaA/betaB). Inhibin is a naturally occurring activin antagonist that consists of an alpha-subunit disulfide-linked to one of the activin beta-subunits, producing inhibin A (alpha/betaA), or inhibin B (alpha/betaB). The development of assays distinguishing between different forms of activins and inhibins, along with knock-in and knock-out models, have provided evidence that the betaA- and betaB-subunits have independent and separate roles physiologically. Additionally, evaluation of ligand-receptor interactions indicates significant differences in receptor affinity between activin isoforms, as well as between inhibin isoforms. In this review we explore the differences between activin/inhibin betaA- and betaB-subunits, including expression patterns, binding properties, and the specific structural aspects of each. From the growing pool of knowledge regarding activins and inhibins, the emerging data support the hypothesis that betaA- and betaB-subunits are functionally differently.

Activin, inhibin and the human breast.

Activins and inhibins are growth factors involved in cell differentiation and proliferation. Human breast tissues such as normal mammary tissue, fibroadenoma, and breast cancer express inhibin and activin mRNA and proteins. Activin A and its binding protein, follistatin, are also present in human milk during the first week of lactation. Using immunohistochemistry, we have observed that the inhibin/activin alpha, betaA, and betaB subunits are present in normal breast tissue regardless of menstrual cycle phase or menopause, as well as in fibrocystic disease, and breast tumors. The mRNAs encoding all three activin/inhibin subunits are expressed in breast carcinoma, fibroadenoma, and normal mammary tissue. The betaA subunit gene expression is higher in either local or metastatic breast carcinoma than in normal tissue. In addition, dimeric activin A is detectable in homogenates of breast cancer tissue at concentrations twice as high as in non-neoplastic adjoining tissue. Recent evidence suggests that some of the activin A produced by breast carcinoma is released into systemic circulation. In women with breast cancer, serum activin A levels are often elevated, and a significant decrease is observed in the first and second days following tumor excision. The role of activin and inhibin as endocrine and/or paracrine factors in the breast is still uncertain. Activin has complex effects on cell growth during branching morphogenesis, but it is generally considered as an inhibitor of cell proliferation as in vitro studies have shown that activin A treatment of breast cancer cells arrests cell growth. Inhibin is generally considered as a tumor suppressor, but its possible role in the breast is less understood.

Shh-dependent differentiation of intestinal tissue from embryonic pancreas by activin A.

The pancreas develops from the endoderm to give rise to ducts, acini and islets of Langerhans. This process involves extracellular signals of the Transforming Growth Factor beta (TGFbeta) family. The aim of this work was to study the effects of activin A, a member of this family, whose potential role in pancreas differentiation is controversial. To this end, we used pancreatic explants from E12.5 mouse embryos. In culture these explants exhibited spontaneous growth, epithelial morphogenesis and endocrine and exocrine differentiation. Exposure to activin A did not affect exocrine or endocrine differentiation. Surprisingly, activin A induced in the explants the appearance of a large contractile structure surrounded by a cylindrical epithelium, a thick basal lamina and a smooth muscle layer. This structure, the formation of which was prevented by follistatin, was typical of an intestinal wall. Consistent with this interpretation, activin A rapidly induced in the explants the mRNAs for fatty acid binding proteins (FABPs), which are markers of the intestine, but not of the pancreas. We also found that induction of the FABPs was preceded by induction of Sonic hedgehog (Shh), a known inducer of intestinal differentiation in the endoderm. Activin B induced neither Shh nor intestinal differentiation. The activin A-mediated intestinal differentiation was blocked by cyclopamine, an inhibitor of Hedgehog signaling, and it was mimicked by Shh. We conclude that activin A does not appear to affect the exocrine or endocrine components of the pancreas, but that it can promote differentiation of pancreatic tissue into intestine via a Shh-dependent mechanism. These findings illustrate the plasticity of differentiation programs in response to extracellular signals in the pancreas and they shed new light on the regulation of pancreas and intestinal development.

Structures of an ActRIIB:activin A complex reveal a novel binding mode for TGF-beta ligand:receptor interactions.

The TGF-beta superfamily of ligands and receptors stimulate cellular events in diverse processes ranging from cell fate specification in development to immune suppression. Activins define a major subgroup of TGF-beta ligands that regulate cellular differentiation, proliferation, activation and apoptosis. Activins signal through complexes formed with type I and type II serine/threonine kinase receptors. We have solved the crystal structure of activin A bound to the extracellular domain of a type II receptor, ActRIIB, revealing the details of this interaction. ActRIIB binds to the outer edges of the activin finger regions, with the two receptors juxtaposed in close proximity, in a mode that differs from TGF-beta3 binding to type II receptors. The dimeric activin A structure differs from other known TGF-beta ligand structures, adopting a compact folded-back conformation. The crystal structure of the complex is consistent with recruitment of two type I receptors into a close packed arrangement at the cell surface and suggests that diversity in the conformational arrangements of TGF-beta ligand dimers could influence cellular signaling processes.

The CEC behaviour of several synthetic peptides related to the activin betaA-betaD subunits.

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.

Molecular cloning and expression of turkey inhibin-alpha and -betaA subunits.

We isolated cDNA encoding turkey inhibin-alpha (tINH-alpha) and -betaA (tINH-betaA) subunits from the turkey ovary using reverse transcription-polymerase chain reaction (RT-PCR). The isolated alpha subunit and betaA subunit included the entire open reading frames encoding 329 and 424 amino acids, respectively. The amino acid sequences of mature tINH-alpha subunit and tINH-betaA subunit (12.6 and 12.9 kDa proteins, respectively), established via DNA sequence analysis, were highly conserved between the chicken and various mammals. Northern blot analysis revealed that the transcripts of tINH-alpha and tINH-betaA subunits were approximately 1.7 and 8.4 kb, respectively. In various stages of follicular development, tINH-alpha mRNA was highly expressed in small white follicles as compared to postovulatory and regressed follicles, whereas tINH-betaA mRNA was predominately expressed in preovulatory F5 follicles.

Total chemical synthesis of human activin beta(A)[12-116] and related large-loop polypeptides.

We report here the synthesis, purification, and characterization of several large polypeptides related to the human activin beta(A) subunit and their cyclic counterparts. In particular, we describe for the first time the total chemical synthesis of a 105-mer polypeptide, des[1-11] activin beta(A), and related large-loop polypeptide, by an optimized solid phase synthetic protocol based on 9-flouroenylmethyoxycarbonyl (Fmoc) chemistry. These studies show that automated chemical synthesis utilizing Fmoc-based solid phase synthetic strategies provides a practical alternative to recombinant DNA technology for the production of activin-related subunits, with the opportunity to rapidly provide different analogues and structural variants for subsequent structure-function and associated biophysical investigations.