Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell.

Follistatin (FS), an activin-binding protein, is a monomer derived from two polypeptide core sequences of 315 (FS-315) and 288 (FS-288) amino acids originated from alternatively spliced mRNA. To define the structural heterogeneity of native FS, we purified six molecular forms of FS from porcine ovaries. Protein chemical analysis revealed that the structural differences among the six isoforms were caused by truncation of the carboxyl-terminal region and/or the presence of carbohydrate chains, resulting in the formation of FS-315, FS-288, and FS composed of 303 amino acids (FS-303) in various forms of glycosylation on the two potential Asn-linked glycosylation sites.

Expression of alpha, beta A and beta B subunits of inhibin or activin and follistatin in rat pancreatic islets.

We first detected the mRNA expression of follistatin and three subunits of inhibin/activin in rat pancreatic islets by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry using anti-follistatin serum (against residues 123-134) revealed that follistatin was localized only in insulin-producing B cells. Although the beta A subunit was detectable in the islets, the immunostainable cell types were completely different with two beta A antisera, i.

The Activin A-Dependent Proliferation of PCC3/A/1 Embryonal Carcinoma Cells in Serum-Free Medium: (Activin/EC/ES cell/Follistatin/Serum-free medium/LIF).

Examination of the growth requirements of murine embryonal carcinoma cells (EC cells) or embryonic stem cells (ES cells) in serum-free medium revealed that PCC3 EC cells required activin A to grow and/or survive in such medium. In the absence of activin A, PCC3 cells began to disintegrate within 3 days under any serum-free conditions examined. P19 and AT805 EC cells grew even in serum-free medium without activin A but their growth rates were slightly facilitated by its addition.

Isolation and characterization of activin receptor from mouse embryonal carcinoma cells. Identification of its serine/threonine/tyrosine protein kinase activity.

The activin receptor protein was isolated from the mouse embryonal carcinoma (EC) cell line P19 by three cycles of affinity chromatography on an activin A-immobilized column. The purified receptor had a specific and high affinity for activins A, AB, and B (Kd = 345 pM), but not for transforming growth factor beta. The purified activin receptor was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting analysis as a single protein of 70 kDa.

Competitive protein binding assay for activin A/EDF using follistatin determination of activin levels in human plasma.

A sensitive and specific protein binding assay for activin A/EDF (activin) was developed using follistatin as a binding protein and [125I] labelled activin as a tracer. As 50% acetonitrile (CH3CN) separated free and follistatin-bound activin, plasma pretreated with an equal volume of CH3CN was used as the assay sample and B/F separation was also done with 50% CH3CN. The recovery of the assay was 85.

Banding profiles of LTR of human endogenous retrovirus HERV-A in 24 chromosomes in somatic cell hybrids.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes. We investigated the distribution of one of these sequences, HERV-A, in 24 human chromosomes by Southern analyses using DNAs from flow-sorted chromosomes or rodent cells carrying a single human chromosome. The results showed that HERV-A is distributed among all human chromosomes and that each chromosome has a specific Southern blot profile.

Paracrine effect of folliculo-stellate cells on the growth factor-like action of activin A in anterior pituitary cultures.

Several studies have shown that pituitary folliculo-stellate (FS) cells exhibit local functions within the pituitary gland. On the other hand, we have shown previously that activin A increases the number of FSH-producing gonadotropes in cultured rat anterior pituitary cells. In this study, we investigated whether FS cells exert an influence on the action of activin A.

Follistatin inhibits activin-induced differentiation of rat follicular granulosa cells in vitro.

The effect of follistatin on activin-induced granulosa cell differentiation was investigated in freshly harvested granulosa cells from diethylstilbestrol (DES)-treated rats. Activin induced a remarkable change in granulosa cellular morphology from elongated fibroblast-like to round cells, which follistatin prevented. Follistatin itself had no influence on the cellular morphology.

Follistatin is a developmentally regulated cytokine in neural differentiation.

Activin acts mitogenically on P19 cells as well as being inhibitory of the differentiation of retinoic acid-treated P19 cells and some neuroblastoma cell lines. Here, we show some lines of evidence that follistatin, an activin-binding protein, is also involved in neural differentiation. Counteracting the activity of activin, addition of follistatin suppresses the anchorage-independent growth of P19 cells in soft agar and stimulates neurite outgrowth of a neuroblastoma cell line, IMR-32 cells.

A monoamine-regulated Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and the maoC gene.

The Klebsiella aerogenes gene maoA, which is involved in the synthesis of monoamine oxidase, was induced by tyramine and the related compounds, subjected to catabolite and ammonium ion repression, and cloned. The nucleotide sequence of the region involved in monoamine oxidase synthesis was determined. Two open reading frames, the maoA gene and a hitherto unknown gene (maoC), were found.

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment.

Evidence for the participation of endogenous activin A/erythroid differentiation factor in the regulation of erythropoiesis.

Activin A/erythroid differentiation factor (EDF) is a human protein that induces differentiation of a murine erythroleukemia cell (the Friend cell). In this study, we demonstrate that endogenous activin A/EDF activity is present in murine bone marrow and spleen. In addition, this activity is secreted by bone marrow and spleen cells in primary culture.

Purification and characterization of high molecular weight forms of inhibin from bovine follicular fluid.

High molecular mass forms [95 kilodaltons (kDa)] of bovine inhibin-A as well as the known forms of intermediate (55 kDa) and low (32 kDa) mass were purified from bovine follicular fluid by ion exchange chromatography on DEAE-Sepharose, immunoaffinity chromatography using a monoclonal antibody directed against bovine 32-kDa inhibin-A, gel permeation HPLC on TSK-gel, and reverse phase HPLC. The 95-kDa inhibin-A had similar suppressive activity on FSH secretion from cultured rat anterior pituitary cells as the 55- and 32-kDa inhibins. There is, however, a possibility that the inhibin activity detected with larger forms may be due to that of the 32-kDa form that results from proteolytic processing during incubation with rat pituitary cells.

Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells.

Follistatin, an activin-binding protein secreted by cultured rat granulosa cells, was shown to associate with the cell surface by affinity labeling with 125I-activin. Addition of follistatin to the cultured cells demonstrated a typical ligand-binding saturation curve, suggesting that granulosa cells have a specific binding site for follistatin. This binding was markedly inhibited by heparin and heparan sulfate, but not by chondroitin sulfates A and C, keratan sulfate, and dermatan sulfate.

Presence of activin (erythroid differentiation factor) in unfertilized eggs and blastulae of Xenopus laevis.

Activin A, a member of the transforming growth factor beta superfamily, has recently been found to have potent mesoderm-inducing activity on isolated early Xenopus animal-cap cells. We measured the activin activity of the Xenopus egg extract by using an erythroid-differentiating test with Friend leukemia cells. The results showed that an activin homologue is, indeed, contained in unfertilized eggs and blastulae of Xenopus laevis in a considerable amount.

Immunohistochemical localization of follistatin in rat tissues.

We have used immunohistochemistry to localize follistatin/activin-binding protein in adult male and female rats. A polyclonal antibody directed against a follistatin peptide (residues 123-134) was used as a specific immunologic probe. Intense and specific follistatin immunoreactivity was evident in spermatogenic cells of seminiferous tubules in the testis.

Characterization of antisera directed against follistatin/activin-binding protein peptides.

An attempt was made to develop immunologic probes directed against follistatin/activin-binding protein (ABP), for use in investigating the distribution of ABP in various tissues. Five oligopeptides corresponding to different regions of the predicted ABP amino acid sequence (peptides 1-12, 28-43, 123-134, 270-281 and 300-315) were synthesized chemically, and coupled to Limulus polyphemus hemolymph hemocyanin. The peptide-hemocyanin conjugates were then used to immunize rabbits, and the immunoresponses were monitored by enzyme-linked immunosorbent assay.

Gene cloning of the maoA gene and overproduction of a soluble monoamine oxidase from Klebsiella aerogenes.

We cloned the structural gene for monoamine oxidase (maoA) from Klebsiella aerogenes into a pKI212 vector in an maoA mutant strain of K. aerogenes. Deletion analysis and complementation tests of the recombinant plasmid showed that the maoA gene was located entirely within a 4.

Follistatin inhibits the mesoderm-inducing activity of activin A and the vegetalizing factor from chicken embryo.

The induction of mesoderm is an important process in early amphibian development. In recent studies, activin has become an effective candidate for a natural mesoderm-inducing factor. In the present study, we show that follistatin, an activin-binding protein purified from porcine ovary, inhibits the mesoderm-inducing activity of recombinant human activin A (rh activin A), which is identical to the erythroid differentiation factor (EDF).

The vegetalizing factor from chicken embryos: its EDF (activin A)-like activity.

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein.

Production of activin-binding protein by rat granulosa cells in vitro.

We have developed an assay method for activin-binding protein, which exploits its high affinity for sulfated polysaccharides. We used this method to investigate the production of activin-binding protein by rat ovarian granulosa cells, in vitro. The production of activin-binding protein by granulosa cells was dependent on the cell density; the maximum was observed at 6 x 10(5) cells/ml.

Activin-binding protein is present in pituitary.

A binding protein for activin was purified from bovine pituitary by affinity chromatography on dextran sulfate-Sepharose CL-4B and activin-Affi-Gel 10. A 52,700-fold purification over the starting crude homogenate was achieved. The purified preparation showed two bands of 36 and 33 kilodalton in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions.

Beta-subunits of equine chorionic gonadotropin and lutenizing hormone with an identical amino acid sequence have different asparagine-linked oligosaccharide chains.

The glycoprotein hormones, equine chorionic gonadotropin (eCG) and lutenizing hormone (eLH), possess a beta-subunit with an identical amino acid sequence. The Asn-linked oligosaccharide chains of eCG beta and eLH beta were quantitatively liberated as tritium-labeled oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4-reduction. Paper electrophoresis in combination with sialidase digestion and solvolytic desulfation indicated that eCG beta contained neutral and sialylated oligosaccharides, while eLH beta contained neutral, sialylated, sulfated, and both sialylated and sulfated oligosaccharides.

Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human x rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched.

A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene.

The structural gene for arylsulfatase (atsA) of Klebsiella aerogenes was cloned into a pKI212 vector in Escherichia coli. Deletion analysis showed that the atsA gene with the promoter region was located within a 3.2-kilobase cloned segment.