Shunichi Shimasaki

Basic Fibroblast Growth Factor (FGF) in the Central Nervous System: Identification of Specific Loci of Basic FGF Expression in the Rat Brain

The expression of basic FGF mRNA, while virtually absent in peripheral tissues, appears to be constitutively expressed in the central nervous system. As such, while it is difficult to detect any mRNA encoding basic FGF in the heart, lung, kidneys, ovaries, liver, or pituitary of rats, the levels are easily detected in brain. A regional analysis of the expression of basic FGF mRNA in brain reveals that it is widely distributed in the cortex (frontal, parietal, and occipital), the hippocampus, hypothalamus, and pons. Only a few loci of basic FGF synthesis are detected by in situ hybridization and include layers 2 and 6 of the medial (cingulate) cortex, the indusium griseum, fasciola cinereum, and field CA2 of the hippocampus. The identification of specific cell populations in the brain, and particularly in the hippocampus, that synthesize basic FGF supports the notion that this potent neurotrophic factor is involved in normal CNS function and that the presence (or absence) of its expression may be linked to the pathogenesis of the neurogenerative diseases characterizing these various loci. The significance of these findings with respect to the regulation of basic FGF expression in peripheral tissue and the central nervous system is discussed.

A homodimer of the β-subunits of inhibin a stimulates the secretion of pituitary follicle stimulating hormone

Summary 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 β-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 β-subunits of inhibin A joined together by disulfide bond(s). The name homo-activin-A is proposed for this substance.

Neuronal nicotinic acetylcholine receptor β‐subunit is coded for by the cDNA clone α4

Acetylcholine receptors (AChRs) with high affinity for nicotine but no affinity for α‐bungarotoxin, which have been purified from rat and chicken brains by immuno‐affinity chromatography, consist of two types of subunits, α and β [1,2]. The β‐subunits form the ACh binding sites [3]. Putative nicotinic AChR subunit cDNAs α3 and α4 have been identified by screening cDNA libraries prepared from rat PC12 cells and rat brain with cDNA probes encoding the mouse muscle AChR α‐subunit. Here we determine the amino‐terminal amino acid sequence of the rat brain AChR β‐subunit by protein microsequencing to be the same as amino acid residues 27–43 of the protein which could be coded by α4. Further, we present evidence consistent with a subunit stoichiometry of α3β2 for this neuronal nicotinic AChR.

Follistatin specifically inhibits pituitary follicle stimulating hormone release invitro

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.

Amino-terminal sequence of a large form of basic fibroblast growth factor isolated from human benign prostatic hyperplastic tissue

Homogenization of human benign prostatic hyperplastic tissue in high ionic strength alkaline buffer containing protease inhibitors resulted in the isolation of a 17,400 molecular weight growth factor. When tissue was homogenized in ammonium sulfate at pH 4.5 without protease inhibitors a smaller, 16,600 dalton, growth factor was isolated. Both growth factors reacted with antisera against synthetic peptides whose sequences corresponded to the amino-terminal (1-12), Internal (33-43) and carboxyl-terminal (135-145) portions of basic fibroblast growth factor (bFGF). This suggested that the smaller growth factor was not a truncated form of (1-146) bFGF and that the larger growth factor may contain additional sequences. Amino-terminal sequencing showed the larger growth factor to have the sequence: Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu-Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly- Ala-Phe-Pro-. These results show that the larger growth factor is an 8 amino acid extended from of (1-146) bFGF and it is likely that the smaller growth factor is a proteolytic cleavage product of the larger growth factor produced during the extraction procedure.

Inhibins and activins

Publisher Summary This chapter discusses the isolation, structure, characterization, and biosynthesis of inhibins and activins. The two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) which regulate the development and maturation of the gonad, are identified. The ability to quantitate FSH and LH in serum revives the inhibin concept, which offers an explanation for the differential regulation of FSH and LH release often observed in humans and experimental animals. Inhibin activity is monitored by an in vitro bioassay using rat anterior pituitary cells in monolayer culture. The message encoding each of the subunits of the inhibins is cloned from porcine and bovine ovarian complementary DNA (cDNA) libraries. DNA sequence analysis shows that the inhibin a subunit is initially synthesized as a large precursor protein with potential N-linked glycosylation sites. The two β subunits are also found to be derived from large polypeptide precursors and cleavage at the processing sites to yield the mature β subunits at the carboxy termini of the precursors. Using the porcine cDNAs as hybridization probes, the human and murine inhibin subunit precursors are also cloned and sequenced.

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.

Fibroblast Growth Factors as Local Mediators of Gonadal Function

Although the presence and effects of many growth factors in gonadal tissues has been established by several investigators (1–4), their physiological function remains, to a large extent, speculative. The main reason behind this problem is the fact that in in vitro assays, growth factors have a tremendous number of biological activities. A case in point is basic fibroblast growth factor, basic FGF (3,4). This mitogen is characterized by its ability to elicit a neovascular response in in vivo assays of angiogenesis (5) and is thought to participate in these processes in the reproductive system (3). Yet these growth factors are also characterized by their capacity to modulate the growth and function of a wide number of cells (6,7). These include granulosa, adrenocortical, endothelial and smooth muscle cells, chondrocytes and fibroblasts, just to name a few. In some instances, basic FGF has no effect on cell proliferation, but only affects differentiated function (8–10). In other cases, its mitogenic activity appears to be the predominant activity (6,7). With the structural characterization of basic FGF, it has become of paramount importance to establish its possible physiological function(s) in tissues where it has been identified. At first glance, its pleiotropic activities might seem to preclude determining a specific function for this molecule; however, the discovery that it is widely distributed and found in almost all tissues suggests that its biological activities are local and thus potentially highly specific for any given environment. On this basis, it seems unlikely that an adrenal-derived FGF plays the function of a wound healing and/or angiogenic factor. More likely it is involved in adrenocortical homeostasis, an activity of the molecule in vitro (11).