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R Guillemin

Publications and source records attributed to R Guillemin.

At least 37 records · Page 2Linked to original sources
PubMed1988

Receptor- and heparin-binding domains of basic fibroblast growth factor.

Two functional domains in the primary structure of basic fibroblast growth factor (FGF) have been identified on the basis of their ability to interact with the FGF receptor, bind radiolabeled heparin, and modulate the cellular response to FGF. Peptides derived from these two functional domains can act as partial agonists and antagonists in biological assays of FGF activity. Peptides related to the sequences of FGF-(24-68)-NH2 and FGF-(106-115)-NH2 inhibit thymidine incorporation into 3T3 fibroblasts when they are stimulated by FGF but have no effect when the cells are treated with either platelet-derived growth factor or epidermal growth factor. They also possess partial agonist activity and can stimulate DNA synthesis when tested in the absence of exogenous FGF. The active peptides have no effect on the binding of epidermal growth factor to its receptor on A431 cells and they can modulate the effects of FGF, but not fibronectin, on endothelial cell adhesion. The results suggest the possibility of designing specific analogs of FGF that are capable of inhibiting the biological effects of FGF.

Amino Acid Sequence↗
PubMed1988

Myelin basic protein present in the acid extracts of rat hypothalami releases insulin and glucagon from isolated rat pancreatic islets.

The insulinotropic and glucagon-releasing activity of acid extracts of rat hypothalami were tested in two bioassay systems using short-time incubation of isolated rat pancreatic islets and a rat insulinoma (RIN) cell line. Release of insulin and glucagon into the incubation medium was measured by radioimmunoassay. The major insulin-releasing and glucagon-releasing activity eluted in a broad zone in Sephacryl S-200 gel filtration in 30% acetic acid corresponding to the molecular weights between approximately 10 and 40 kD. The highest activity was eluted in a zone corresponding to 14 kD and was purified to homogeneity by means of two steps of reversed-phase HPLC. Amino acid analysis and SDS polyacrylamide gel electrophoresis indicated that the biologically active material was the rat small (myelin) basic protein characterized previously by Dunkley & Carnegie (1974). The purified rat hypothalamic material showed insulinotropic and glucagon-releasing activity indistinguishable from that of purified porcine myelin basic protein, and lost its insulinotropic activity when incubated with anti-myelin basic protein immunosorbent. We conclude that the major insulin-releasing and glucagon-releasing activity in acid extracts of the high-molecular-weight fractions of rat hypothalami is myelin basic protein.

Amino Acids↗
PubMed1987

Follistatin specifically inhibits pituitary follicle stimulating hormone release in vitro.

Two forms of purified follistatin, a single-chain polypeptide of mol wt 35,000 (35 Kd) protein, and a related molecule of mol wt 32,000 (32 Kd), which differs from the 35 Kd form in glycosylation or carboxyl terminal truncation, specifically inhibit the release of immunoreactive FSH by primary cultures of rat pituitary cells. Both forms of follistatin and inhibin-A give similar dose-response curves, with identical slopes and maximal effects, suggesting that they may all act through the same mechanism on the pituitary cells. The median effective dose (ED50) of each of the follistatins is 6.2-7.3 ng/ml (1.8 x 10(-10) M), which corresponds to approximately 1/3 of the potency of inhibin. The effect of 35 Kd or 32 Kd follistatin is highly specific for suppressing the release of immunoreactive FSH since there is no demonstrable concomitant effect on the secretion of other pituitary hormones. The effect of follistatins, like that of inhibins, is different from that of the hypothalamic hypophysiotropic factors, requiring greater than or equal to 18 h of incubation in a pituitary monolayer culture system to demonstrate. Coincubation of inhibin and follistatin shows an additive effect in the suppression of FSH release. Pituitary cells exposed to follistatin have significantly less depletion of intracellular FSH (0.01) than those treated with inhibin, indicating that follistatin may act primarily on the suppression of FSH release rather than on both release and synthesis of FSH, as is the case with inhibin.

Animals↗
PubMed1987

Immunohistochemical detection of inhibin in the gonad.

Antiserum to inhibin was produced in rabbits by immunization with a synthetic [Tyr30]alpha-chain(1-30)NH2 fragment of porcine inhibin coupled to bovine serum albumin, and the elicited antiserum was used in conjunction with the avidin-biotin immunoperoxidase procedure to localize inhibin-reactive cells in various rat tissue preparations. In the testes, only the Sertoli cells revealed immunoreactivity with the antiserum. Intense staining was also observed in ovarian follicular granulosa cells but not in the theca layer outside the basement membrane. In addition, the luteal cells in the corpus luteum were also stained by the antiserum. The positive staining in the gonadal tissues could be blocked completely by pre-adsorbing the serum with either the synthetic peptide or native inhibin. Immunostaining was not detected in brain, pituitary, thymus, stomach, pancreas, kidney and adrenal section, thus confirming that inhibin is a polypeptide originating only from specific cells of the gonad.

Animals↗
PubMed1987

Isolation and partial characterization of basic fibroblast growth factor from bovine testis.

A basic fibroblast growth factor (FGF) has been purified to homogeneity from bovine testis, using ammonium sulfate precipitation of the crude extract followed by three chromatographic steps, involving cation-exchange, heparin-Sepharose, and reversed-phase HPLC. Gas-phase sequence analysis showed the amino-terminal amino acid sequence of the isolated polypeptide as His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-, which is identical to the amino-terminal of the (16-146) fragment of basic FGF previously characterized from corpus luteum, adrenal, and kidney. The purified FGF was shown to have the same biological activity as that of basic FGF (1-146). This finding suggests that basic FGF is present in testis and may act as a local regulator of testicular function. In addition, testicular FGF might play an important role in spermatogenesis and/or the development of testis.

Amino Acid Sequence↗
PubMed1987

Secretion of follicle-stimulating hormone and production of inhibin are reciprocally related.

The production of inhibin in cultured granulosa cells from immature hypophysectomized, estrogen-treated rats and Sertoli cells from normal animals was determined by a specific radioimmunoassay using an antiserum against a synthetic replicate of [Tyr30]inhibin alpha-chain-(1-30). The amount of immunoreactive inhibin detected in the spent media of these cells is in proportion to the density of cells plated and the concentration of exogenously added follicle-stimulating hormone (FSH). In the presence of the estrogen precursor androstenedione (10(-7) M), FSH, but not luteinizing hormone, produced a dose-dependent increase in inhibin during 2-day culture of granulosa cells. In the absence of the estrogen precursor, similar but somewhat diminished inhibin production in responding to FSH was observed. Exogenously added estrogen potentiated the FSH-mediated release of inhibin in the absence of androstenedione. Neither androstenedione nor estradiol added to the cultured Sertoli cells had effect on inhibin production. A preparation of pure inhibin isolated on the basis of an in vitro bioassay and characterized chemically specifically suppressed serum FSH but not luteinizing hormone, when it was injected (24 micrograms per injection, two injections) into acutely ovariectomized rats. Thus, inhibin secreted by the granulosa and Sertoli cells specifically suppresses the secretion of pituitary FSH, and in turn FSH is primarily responsible for the inhibin production in these gonadal cells, as in a classical negative-feedback relationship.

Androstenedione↗
PubMed1987

Isolation and partial characterization of follistatin: a single-chain Mr 35,000 monomeric protein that inhibits the release of follicle-stimulating hormone.

A Mr 35,000 protein with follicle-stimulating hormone release-inhibitory activity was isolated from porcine ovarian follicular fluid by heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and multiple steps of high-performance liquid chromatography. The isolated molecule is highly enriched in cysteines and is composed of a single polypeptide chain. In addition, it has no sequence homology with the previously characterized follicular fluid inhibins, which are heterodimeric proteins of Mr 32,000 with follicle-stimulating hormone release-inhibiting activity. This protein specifically inhibits the basal secretion of follicle-stimulating hormone, but not that of luteinizing hormone, in the rat anterior pituitary monolayer culture system with a half-maximal effective dose of 2.5-6.0 ng/ml. Another form of the molecule of Mr 32,000 present in much lower concentration in follicular fluid was also isolated. It may differ from the Mr 35,000 form in glycosylation or carboxyl-terminal truncation. We suggest that this compound be called "follistatin" to signify its structural difference from inhibin.

Amino Acids↗
PubMed1987

Structural characterization of follistatin: a novel follicle-stimulating hormone release-inhibiting polypeptide from the gonad.

Follistatin, a novel, single chain, glycosylated polypeptide bearing no homology with previously characterized inhibins but exhibiting potent and specific pituitary FSH-release inhibition has been structurally characterized by protein microsequencing, cDNA cloning, and DNA sequencing. Two populations of clones differing in their 3'-untranslated sequences were found to encode a 344 amino acid precursor protein and an identical but carboxyl terminal truncated 317 amino acid precursor, respectively. Additionally, one clone, FS18, contained two introns and probably resulted from reverse transcription of heterogeneous nuclear RNA during cDNA library construction. Follistatin is unusually cysteine-rich, containing 36 cysteines in the mature coding sequence of 315 amino acids and an extremely acidic carboxyl terminal region, FS(292-304), comprised of Glu-Asp-Thr-Glu-Glu-Glu-Glu-Glu-Asp-Glu-Asp-Gln-Asp which probably resides outside a tightly cross-linked protein sphere. The heparin-binding ability of follistatin can probably be ascribed to the basic region specified by FS(75-86), Lys-Lys-Cys-Arg-Met-Asn-Lys-Lys-Asn-Lys. Overall, follistatin is organized into three homologous domains, FS(66-135), FS(139-210), and FS(216-287) containing 70, 72, and 72 amino acids, respectively, which show a 52% homology among themselves and a 57% homology with the 56 amino acid human pancreatic secretory trypsin inhibitor protein when aligned for maximum homology.

Amino Acid Sequence↗
PubMed1986

Purification and partial characterization of a mitogenic factor from bovine liver: structural homology with basic fibroblast growth factor.

Two mitogenic peptides in bovine liver extract were purified to apparent homogeneity by monitoring the purification steps with two in vitro bioassays; one based on stimulation of adult bovine aortic arch endothelial cell proliferation and the other incorporation of [3H]thymidine to mouse fibroblast 3T3 cells. The purification procedure involved cation-exchange chromatography followed by affinity chromatography on heparin-Sepharose and two steps of reversed-phase HPLC. The purified material showed the same biological activity as pituitary basic fibroblast growth factor (FGF). Amino acid analyses of the purified mitogen yielded a similar, but not identical composition to that of bovine pituitary basic FGF(1-146) reported previously. Gas-phase microsequencing identified two sequences in equal amounts in the purified preparation. Furthermore, the sequencing results are in accord with the theoretical data obtained when two truncated forms of basic FGF, corresponding to FGF(12-146) and (16-146), are being sequenced simultaneously. Basic FGF(12-146) is a novel truncated form of basic FGF which has not been isolated before although the (16-146) fragment has been found previously in kidney, corpus luteum, and adrenal. SDS-PAGE analysis could not separate the two forms and showed that both migrated as a protein of about 15,100 daltons, which is slightly smaller than intact basic FGF(1-146) (16,200 daltons). These results, taken together, indicate that at least some of the mitogenic activity in liver may be derived from basic FGF-related polypeptides.

Amino Acid Sequence↗
PubMed1986

A homodimer of the beta-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone.

A 24,000 Dalton protein with follicle stimulating hormone (FSH)-releasing activity, named activin, has been characterized previously from porcine follicular fluid as a heterodimer composed of the beta-subunits of inhibins A and B linked by disulfide bond(s) [Ling et al. (1986) Nature, in press]. In this paper we report the isolation of another 24,000 Dalton protein with FSH-releasing activity from porcine follicular fluid, using successive steps of heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and four steps of reversed-phase HPLC, followed by preparative sodium dodecyl-sulfate-polyacrylamide gel electrophoresis chromatography. Based on the molecular weight of the isolated molecule and its deduced NH2-terminal sequence, we propose that this second FSH-releasing substance present in porcine follicular fluid is a homodimeric protein composed of two beta-subunits of inhibin A joined together by disulfide bond(s). The name homo-activin-A is proposed for this substance.

Amino Acid Sequence↗
PubMed1986

Isolation of an amino terminal extended form of basic fibroblast growth factor.

Extraction of bovine pituitaries in the presence of enzyme inhibitors (2 mM PMSF, 2 mM sodium tetrathionate, 15 microM pepstatin A, and 1 mM EDTA) resulted in the isolation of two distinct forms of basic fibroblast growth factor. Partial characterization of both molecules showed one form to be identical to basic FGF(1-146) which has already been reported by our laboratory. The second form was estimated by SDS-PAGE to have a molecular weight of 17,000 Daltons which is slightly larger than that of basic FGF(1-146). Amino acid analysis shows the presence of 8 new residues more than basic FGF(1-146) which accounts for the difference in molecular weight. Gas-phase sequencing of this molecule indicated that it bears a blocked amino terminus. Furthermore, this higher molecular weight form of basic FGF did not show immunoreactivity with antibodies specific for the amino terminus of basic FGF(1-146) but cross reacted with antibodies generated against midportion fragments of basic FGF(1-146), indicating that the molecule is amino terminally extended. Like basic FGF(1-146), the molecule is a potent mitogenic factor for vascular endothelial cells. Taken together these results demonstrate the existence of a precursor form of basic FGF which is extended by 8 residues at the amino terminus with the first residue being blocked.

Amino Acids↗
PubMed1986

Inhibin and beta type transforming growth factor (TGF beta) have opposite modulating effects on the follicle stimulating hormone (FSH)-induced aromatase activity of cultured rat granulosa cells.

We have recently observed that attomolar concentration of exogenously added TGF beta, a molecule structurally related to inhibin, can stimulate the basal secretion of FSH in a pituitary cell culture. Inhibin purified from porcine follicular fluid antagonizes this activity of TGF beta. To understand further the homeostatic regulatory properties of inhibin and TGF beta we have investigated whether the aromatase activity of ovarian granulosa cells is also subject to intra-ovarian modulation by these peptides. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for 2 days with androstenedione (10(-7) M) as a substrate, oFSH (2 ng), and different amounts of TGF beta or inhibin. Basal estrogen secretion was negligible and remained unaffected by treatment with purified TGF beta or inhibin (10 ng/ml), whereas treatment with oFSH (2 ng/ml) produced a 100-fold increase in estrogen accumulation. The concurrent application of increasing concentrations (10 pg-10 ng/ml) of TGF beta produced dose-dependent increments in the FSH-stimulated accumulation of estrogen with a ED50 of 0.3 +/- 0.02 ng/ml. On the other hand, concurrent incubation of FSH with inhibin ranging from 10 pg to 10 ng/ml decreases the FSH-mediated estrogen secretion. TGF beta antagonizes the inhibition of inhibin on aromatase activity. These findings suggest that inhibin and TGF beta, two closely related molecules, play novel and opposite roles in modulating the follicular functions.

Androstenedione↗
PubMed1986

Type beta transforming growth factor (TGF-beta) is a potent stimulator of the basal secretion of follicle stimulating hormone (FSH) in a pituitary monolayer system.

In view of striking similarities between TGF-beta and inhibin, we investigated the possibility that TGF-beta might modulate pituitary hormone release in vitro. Long term incubations of beta transforming growth factor (TGF-beta) with rat anterior pituitary cells for 48 hr stimulates the basal secretion of FSH in a dose-dependent manner. The secretion of LH, TSH, GH, ACTH and PRL is not modified by TGF-beta. The minimal effective concentration of TGF-beta is 10 pg/ml (less than 500 attomolar) and is dose dependent over a range from 1 pg to 10 ng/ml. Treatment of cells with TGF-beta for short incubation times (4 hr) in assays similar to that used for hypophysial releasing factors is not effective, indicating that TGF-beta acts through a cellular mechanism distinct from that of LRF. Inhibin-A, recently characterized on the basis of its capacity to specifically inhibit the secretion of FSH in the 48 hr bioassay system inhibits the stimulatory effect of TGF-beta on FSH-release. Analyses of the dose response curves indicate that the interaction occurs in a typical non-competitive manner. The results suggest that a TGF-beta-like molecule, present in follicular fluid, may be responsible for the FSH-releasing activity ("anti-inhibin" activity) observed by us and others during the process of isolating inhibin from follicular fluids. They also suggest an important role for inhibin and the TGF-beta related molecules in modulating pituitary gonadotropin release.

Animals↗
PubMed1986

Basic fibroblast growth factor induces angiogenesis in vitro.

Fibroblast growth factors (FGFs) are potent mitogens for vascular and capillary endothelial cells in vitro and can stimulate the formation of blood capillaries (angiogenesis) in vivo. A crucial event in this process is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. Using a recently developed in vitro model of angiogenesis, we show here that highly purified basic pituitary FGF can induce capillary endothelial cells to invade a three-dimensional collagen matrix and to organize themselves to form characteristic tubules that resemble blood capillaries. We also show that basic FGF concomitantly stimulates endothelial cells to produce a urokinase-type plasminogen activator, a protease that has been implicated in the neovascular response. The results demonstrate that basic FGF can stimulate processes that are characteristic of angiogenesis in vivo, including endothelial cell migration, invasion, and production of plasminogen activator.

Animals↗
PubMed1986

Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.

Basic fibroblast growth factor (FGF) has been found to increase neuronal survival and neurite extension in a highly purified population of fetal rat hippocampal neurons under well-defined serum-free cell culture conditions. In the presence of FGF, neuronal survival after 7 days in culture on a simple plastic substrate is increased 4-fold, to 54% of the initial population. Survival is increased 2-fold to 40% on polyornithine-laminin. When FGF was bound to plastic or heparin substrates, neurite outgrowth was significantly increased to lengths comparable to those seen with laminin; however, FGF produced no further increase in neurite outgrowth on laminin. Half-maximal survival was observed at FGF concentrations of about 15 pg/ml (1 pM); half-maximal process outgrowth occurred at about 375 pg/ml (20 pM). The responsive cells were identified as neurons by their labeling with tetanus toxin and by antibodies to neurofilaments and to the neuron-specific enolase. Astrocytes, identified by the presence of glial fibrillary acidic protein, constituted about 10% of cells present at 1 week both in the presence and in the absence of FGF. These results strongly suggest that, in addition to its known mitogenic effects on nonneuronal cells, FGF possesses neurotrophic activity for hippocampal neurons.

Animals↗
PubMed1986

Immunohistochemical evidence that growth hormone-releasing factor (GRF) neurons contain an amidated peptide derived from cleavage of the carboxyl-terminal end of the GRF precursor.

Antisera were raised against synthetic replicates of the carboxyl-terminal (C-terminal) fragment of the precursor to human GH-releasing factor (GRF) (pre-proGRF) whose structure was predicted from the complementary DNA cloned from a pancreatic tumor. These antisera were used along with antisera to human GRF itself to search for the presence of related molecules in the human hypothalamus, with an immunohistochemical approach. The antisera to pre-proGRF that recognize specifically the C-terminal amidated form of pre-proGRF stain GRF neurons in their cell bodies, fibers, and nerve endings that are in contact with portal capillaries of the median eminence. Antisera against the nonamidated form of the molecule did not give any staining in the hypothalamus. These results strongly suggest that human hypothalamic GRF derives from a precursor immunologically related (and probably identical) to the tumorous one and that this precursor is cleaved inside GRF cell bodies to give, in addition to the GRF-44-NH2 a second amidated peptide, the C-terminal pre-proGRF that is transported distally to nerve endings and most probably coreleased with GRF into portal capillaries.

Adult↗