Biomedical subjects

N Ling

Publications and source records attributed to N Ling.

At least 109 records · Page 6Linked to original sources

PubMed1992

Expression of inhibin beta A and beta B, follistatin and activin-A receptor messenger ribonucleic acids in the rat seminiferous epithelium.

The expression of inhibin beta A and beta B subunits, follistatin, and activin-A receptor messenger RNA (mRNAs) in different stages of rat seminiferous epithelial cycle was analyzed by in situ hybridization in order to understand their role in the regulation of spermatogenesis. Inhibin beta A mRNA was expressed in Sertoli cells in a highly stage-specific manner. The mRNA levels started to accumulate in Sertoli cells at stage VIII of the cycle and were highly expressed during stages IX-XI. Follistatin mRNA expression was identical to that of inhibin beta A, while inhibin beta B mRNA was maximally expressed in Sertoli cells at stages XIII-III. Low expression was found in stages VII-VIII. Activin-A receptor mRNA was localized mainly in spermatogenic cells. Maximal expression was seen in late primary spermatocytes at stages XIII-XIV and in early round spermatids at stages I-IV. A low even expression by Sertoli cells was also seen. Inhibin beta A and follistatin mRNAs were coexpressed in stage IX-XI Sertoli cells, suggesting close interplay between these molecules. The pattern of inhibin beta B mRNA expression was similar to that of inhibin alpha-mRNA. Localization of activin-A receptor mRNA in spermatogenic cells suggests that activin may influence meiotic divisions and early spermiogenesis.

Activin Receptors↗

PubMed1991

Site-specific mutagenesis of human follistatin.

Follistatin is a monomeric protein originally discovered in ovarian follicular fluid as a suppressor of pituitary follicle-stimulating hormone (FSH) secretion, and later identified as a binding protein for activin. To explore the role of the Asn-linked carbohydrate chains on the follistatin molecule in regard to the inhibition of FSH secretion and activin binding ability, site-specific mutations were introduced at either or both of the two potential Asn-linked glycosylation sites of human follistatin with 315 amino acids (hFS-315). The three types of follistatin mutants were expressed individually in Chinese hamster ovary cells. When tested for their ability to inhibit FSH secretion and to bind activin, each mutant was found to have a similar property as the non-mutated recombinant hFS-315, suggesting that glycosylation of the follistatin molecule has no effect in these functions. However, a two amino acid insertion in between the second and the third amino acid residues in hFS-315 caused the resulting compound to lose completely its inhibitory activity on FSH secretion from the pituitary as well as its binding ability to activin. This finding suggests that the amino-terminal region of the follistatin molecule is critical for both of these functions.

Activins↗

PubMed1991

Brain corticotropin-releasing hormone increases arousal in stress.

The effect of restraint stress on pentobarbital-induced sleeping time was examined in rats. Restraint for 60 and 75 min significantly shortened pentobarbital-induced sleeping time. The shortening of sleeping time by restraint was completely reversed by intracerebroventricular (i.c.v.) administration of alpha-helical CRH(9-41), a corticotropin-releasing hormone (CRH) receptor antagonist. In conjunction with our previous finding that i.c.v. administration of CRH shortens pentobarbital-induced sleeping time, the results suggest that restraint stress increases arousal through brain CRH.

Animals↗

PubMed1991

Identification of five different insulin-like growth factor binding proteins (IGFBPs) from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human.

Five different insulin-like growth factor binding proteins (IGFBPs) were isolated from adult rat serum using gel filtration, ligand affinity chromatography, and two steps of reversed-phase high performance liquid chromatography. Three of them were identified as IGFBP-2, -3, and -4 by their amino-terminal amino acid sequences. One of the remaining two proteins was the rat homologue of the partially characterized IGFBP isolated originally from human cerebrospinal fluid, while the other appeared to be a novel member of the IGFBP family. IGFBP-1 was not found in the adult rat serum under our experimental procedures. cDNAs encoding the novel IGFBP were isolated and characterized from a rat ovary and a human placenta library. The mature protein predicted for both species contained 252 amino acids including 18 cysteines that were located in the homologous positions as IGFBP-1, -2, -3, and -4. We propose to name this protein IGFBP-5. Northern analysis of IGFBP-5 mRNA in rat tissues demonstrated that transcription of this gene is highly active in kidney, although the mRNA was detectable in all tissues examined. Alignment of the amino acid sequences of the five rat IGFBPs revealed a 47-60% similarity, indicating that their individual genes diverged from a single ancestral gene by successive gene duplication in a short time frame during evolution. The chromosomal localizations of IGFBP-1, -2, -3, -4, and -5 genes in human have been determined using polymerase chain reaction on somatic cell hybrid DNAs of human and hamster, and the results showed that they were located on chromosomes 7, 2, 7, 17, and 5, respectively.

Amino Acid Sequence↗

PubMed1991

In vitro release of growth hormone-releasing factor (GRF) from the hypothalamus: somatostatin inhibits GRF release.

The manner of release of growth hormone-releasing factor (GRF) from the rat hypothalamus was studied in a perifusion system using a highly sensitive radioimmunoassay for rat GRF. The recovery of GRF in this system was 50-60%. The release of GRF from the rat hypothalamic blocks was almost stable for 20-240 min after the start of the perifusion and was stimulated by depolarization induced by high K+ concentration. The release of GRF was inhibited by somatostatin at concentrations of 10(-11) to 10(-8) M with maximum inhibition to 52.5% of the basal release at a concentration of 10(-9) M. These results suggest that this system is useful in studying the regulatory mechanism of GRF release and that, in addition to its action on the pituitary, somatostatin appears to act at the level of the hypothalamus in inhibiting GRF release in the regulation of GH secretion.

Animals↗

PubMed1991

Phosphorylation of basic fibroblast growth factor by purified protein kinase C and the identification of a cryptic site of phosphorylation.

We have further characterized the protein kinase C (PK-C) dependent phosphorylation of basic fibroblast growth factor (FGF). Intact recombinant basic FGF and a series of ten peptide fragments of basic FGF were phosphorylated by PK-C and the products were analyzed by SDS-PAGE and autoradiography. As expected, peptide fragments containing the known site of phosphorylation (Ser64) are substrates for phosphorylation. Surprisingly however, peptides containing the receptor binding domain of the mitogen [basic FGF(106-115)] are also phosphorylated. An examination of this sequence reveals the presence of a consensus sequence (Ser108-Ala109-Lys110) that mediates the reaction. Accordingly, all peptides that contain the core amino acids basic FGF(106-111) are substrates for phosphorylation. Peptide mapping of basic FGF confirms that Ser64 is the primary site of phosphorylation, suggesting that Ser108 is a cryptic consensus sequence. Because basic FGF is metabolized to sequence specific fragments after its binding and internalization into target cells, this cryptic site may in fact be phosphorylated in vivo.

Animals↗

PubMed1991

The effects of chronic central administration of corticotropin-releasing factor on food intake, body weight, and hypothalamic-pituitary-adrenocortical hormones.

The effects of chronic central administration of corticotropin-releasing factor (CRF) on food intake, body weight, and hypothalamic-pituitary-adrenocortical hormones were investigated in rats. The infusion of ovine CRF at doses of 0.3 and 1.0 microgram/h continuously induced decrease in food intake and a suppression of body-weight gain for 7 days. The inhibition of body weight gain induced by CRF could not be accounted for solely by a decreased food intake since the suppression of body-weight gain in CRF-infused rats was significantly greater than that observed in rats which received the same amount of food as the CRF-infused rats. The content of proopiomelanocortin (POMC) -derived peptides in the anterior lobe of the pituitary as well as the plasma levels of ACTH and corticosterone (B) were significantly elevated in CRF-treated rats, and the CRF content in the hypothalamus was significantly decreased. These results suggest that chronic intracerebroventricular (icv) administration of CRF stimulates the synthesis and secretion of POMC-related peptides in the pituitary and suppresses food intake accompanied by inhibition of body weight gain. The results are similar to clinical and laboratory findings observed in patients with stress-induced anorexia.

Adrenocorticotropic Hormone↗

PubMed1991

Interleukin-1 inhibits stress-induced gastric erosion in rats.

The effect of interleukin (IL)-1 on the occurrence of stress-induced gastric erosions was examined in rats. The intracerebroventricular (icv) administration of IL-1 beta significantly inhibited the occurrence of water-immersion restraint stress-induced gastric erosion at doses of 200 ng, 500 ng and 1 microgram, whereas the intravenous (iv) administration of IL-1 beta altered the occurrence of gastric erosion only at a dose of 1 microgram. The inhibitory effect of IL-1 alpha icv administered on the occurrence of gastric erosion was found only at a dose of 1 microgram. The inhibitory effect of IL-1 beta icv administered on the occurrence of stress-induced gastric erosion was not influenced by icv administration of alpha-helical CRF(9-41), a corticotropin-releasing factor (CRF) receptor antagonist. Indomethacin completely blocked the inhibitory action of IL-1 beta icv administered on stress-induced gastric erosion. It is concluded from these results that IL-1 acts mainly in the central nervous system to inhibit the occurrence of stress-induced gastric erosion and that the IL-1 beta-induced inhibition of gastric erosion is mediated by prostaglandin in a manner that is independent of brain CRF.

Animals↗

PubMed1991

Stage-specific cellular regulation of inhibin alpha-subunit mRNA expression in the rat seminiferous epithelium.

To find out the local regulation of inhibin production and its possible paracrine role in the seminiferous epithelium, inhibin alpha mRNA levels were measured in sequential 1 mm segments of rat seminiferous tubules accurately staged by transillumination technique. Highest levels were found at stages XIV-I-IV of the cycle, and lowest at stages VI-VIIb of the cycle. When dividing spermatogonia were selectively destroyed by 3 Gy of high-energy X-irradiation, stage-specific inhibin alpha mRNA levels remained unchanged until 26 and 38 days after irradiation when stages VII and VIII of the cycle showed 6- and 4-fold increases during a selective reduction of pachytene spermatocyte and round spermatid numbers, respectively. The results suggest that these cells at a strictly stage-specific fashion have a paracrine inhibitory effect on Sertoli cell inhibin alpha gene expression. Inhibin alpha mRNA level also correlates closely to the follicle-stimulating hormone-stimulated cAMP production during the cycle of the seminiferous epithelium, but does not seem to have a correlation to spermatogonial DNA synthesis.

Animals↗

PubMed1991

Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6).

Six different insulin-like growth factor binding proteins (IGFBPs) have been identified by molecular cloning of their cDNAs from rat and human tissues and designated as IGFBP-1, -2, -3, -4, -5 and -6. The total number of amino acid residues for the mature rat BPs ranges from 201 for IGFBP-6 to 270 for IGFBP-2, while the human homologs range from 216 for IGFBP-6 to 289 for IGFBP-2. Except for IGFBP-6, all rat and human IGFBPs contain 18 homologous cysteines; twelve are located at the N-terminal and span approximately one-third of the total amino acid sequence, while the remaining six are distributed at the C-terminal and span the last one-third of the protein sequence. Both rat and human IGFBP-4 possess two extra cysteines at the mid-region of the molecule. By contrast, rat and human IGFBP-6 contain only 14 and 16 cysteines, respectively. Absence of the two and four cysteines in the N-terminal region in the human and rat IGFBP-6 resulted in the deletion of the invariant Gly-Cys-Gly-Cys-Cys sequence which is present in all the other five IGFBPs. Both rat and human IGFBP-3 possess multiple N-linked glycosylation sites at the mid-region of the molecule, which accounts for their apparent molecular size being larger than the calculated molecular weight, based on the amino acid sequence. One potential N-linked glycosylation site is located at the mid-region of rat and human IGFBP-4, whereas only human but not rat IGFBP-6 possesses one N-linked glycosylation site at the extreme C-terminal of the molecule. An RGD sequence is found in the C-terminal of IGFBP-1 and -2. In this short review, updated information on the structural identification and molecular cloning of the six IGFBPs will be presented. In addition, the potential regulation of the BPs at the transcriptional and translational levels will be discussed.

Amino Acid Sequence↗

PubMed1991

Ovarian intrabursal administration of insulin-like growth factor-binding protein inhibits follicle rupture in gonadotropin-treated immature female rats.

The effects of an insulin-like growth factor-binding protein (IGF-BP) on rat follicular function were examined by using the technique of ovarian intrabursal (IB) injection. Immature female rats were injected with 15 IU of eCG followed immediately with IB injections of 4 micrograms IGF-BP3 (right ovary) and vehicle (left ovary). Forty-eight hours later, the same animals were either killed (eCG-treated group) or injected with 1 microgram of hCG as an ovulatory stimulus. These animals were killed 24 h later (eCG/hCG-treated group). Intrabursal administration of IGF-BP3 inhibited ovulations in the eCG/hCG-treated rats by 55% when compared with the contralateral vehicle-treated ovary (p = 0.01). Examination of the ovaries exposed to IGF-BP3 revealed the presence of unruptured follicles containing a matured oocyte and a disintegrated basement membrane, in addition to normal follicles and corpora lutea. In contrast, IB injection of IGF-BP3 had no effect on ovarian weights or circulating estradiol concentrations in the eCG-treated animals, and the ovaries appeared to be morphologically normal. Ligand blotting experiments using [125I]-labeled insulin-like growth factor I revealed that granulosa cells obtained from both untreated and eCG-treated rats synthesized and secreted two IGF-BPs of Mr 35,000 and 30,000. Equine chorionic gonadotropin treatment reduced the amount of the 30,000 Mr form of IGF-BP. These data suggest that locally produced ovarian IGF-BPs may modulate follicle functions in vivo.

Animals↗

PubMed1991

Cellular origins of serum complement receptor type 2 in normal individuals and in hypogammaglobulinaemia.

A soluble form of complement receptor 2 (sCR2) is found in normal human serum. Amounts present are about 30-90 ng/ml, which is of the same order as reported for soluble CR1. Although B cells express surface CR2 and are the main peripheral blood source of sCR2 they do not appear to be the major tissue source of serum sCR2. Serum levels of sCR2 of patients with hypogammaglobulinaemia were not significantly different from those of normal individuals even in the case of two brothers with Bruton's X-linked agammaglobulinaemia (XLA) lacking (CD19+) B cells. On gel filtration through Sephacryl S-300 the sCR2 from XLA serum behaved exactly like sCR2 from normal serum or sCR2 affinity purified from cell supernates of a B lymphoblastoid line or from the T-ALL line MOLT-4. In all cases a single peak appeared at the same point in the chromatogram. Possible alternative sources of serum sCR2 are follicular dendritic cells (FDC) which are known to express CR2 strongly and T6+ lymphocytes within the thymus. Peripheral T cells from adults have not been reported to express CR2. However, investigation showed that cells from the Bruton's XLA cases produced small amounts of sCR2 in culture and although no CD21 was detected on the surface of the mononuclear cells by flow cytometry, the more sensitive direct antibody rosette test readily detected CD21. Further studies showed that non-B cells from control samples of cord blood or blood of young children also weakly expressed CD21.

Agammaglobulinemia↗

PubMed1991

Solid phase synthesis of human growth hormone-releasing factor analogs containing a bicyclic beta-turn dipeptide.

Three analogs derived from the N-terminal 29-residue fragment of human growth hormone-releasing factor (hGRF) which contained a bicyclic beta-turn dipeptide (BTD) at 7-8, 8-9, and 9-10 positions were synthesized by solid phase methodology to ascertain if the beta-turns are important for the biological activity of hGRF and also to show the applicability of the BTD unit to solid phase synthesis. All three analogs were obtained in good yield and purity indicating that the BTD unit can be used in the usual condition of solid phase synthesis. The capacity of these analogs to release growth hormone (GH) was tested in an in vitro bioassay using rat anterior pituitary cells. All three BTD-containing analogs showed the same maximal GH secretion with parallel dose-response curves to that of hGRF(1-29)NH2, except their relative potencies were very low.

Amino Acid Sequence↗

PubMed1991

[Leu27] insulin-like growth factor II is highly selective for the type-II IGF receptor in binding, cross-linking and thymidine incorporation experiments.

Insulin-like growth factor-II (IGF-II) is able to interact with three different receptors: the type-I and type-II IGF receptors, and the insulin receptor, although with a lower affinity. This cross reactivity obscures the mechanisms via which the biological activities of IGF-II are mediated. We have synthesized an IGF-II analog, [Leu27]IGF-II, that is highly selective for the type-II IGF receptor. Receptor binding experiments demonstrate a high affinity for the type-II IGF receptor, analogous to synthetic (syn) and recombinant (rec) IGF-II, but no affinity for the type-I IGF and the insulin receptor at concentrations up to 50 and 200 ng/ml, respectively. The lack of affinity for these two receptors is confirmed by biological studies which demonstrated that this analog, in contrast with synIGF-II, did not stimulate [3H]thymidine incorporation in Balb/c 3T3 cells. [Leu27]IGF-II opens new ways to identify which actions of IGF-II are mediated via the type-II receptor and which are due to cross reactivity with the type-I IGF or the insulin receptor.

Animals↗

PubMed1991

In vivo comparison of the follicle-stimulating hormone-suppressing activity of follistatin and inhibin in ovariectomized rats.

The present study was performed to compare and contrast the effects of two gonadal polypeptides, inhibin and follistatin, on ovariectomy-induced hypersecretion of FSH and LH. Ovariectomies were performed 1 week before study. Follistatin was purified from porcine follicular fluid, and human inhibin A was produced by recombinant DNA technology. On the day of study, a blood sample was taken from intraatrial cannulae inserted on the previous day, the materials were injected, and additional blood samples were taken at various times thereafter. Serum FSH and LH levels were determined by RIA. Both follistatin and inhibin exhibited dose-dependent suppression of circulating FSH but not LH levels, with initial decreases in FSH levels by both materials observed between 2-4 h post injection. Maximal suppression of FSH by each polypeptide occurred between 4-6 h depending on dose. Based on the dose-response relationships, it was determined that inhibin was approximately five times as potent as follistatin in suppressing FSH release. However, despite the greater biopotency of inhibin than follistatin, the duration of action of even the highest dose of inhibin (50 micrograms) was between 4-9 h, whereas the duration of FSH-suppressing activity by the two highest doses of follistatin (40 and 80 micrograms) was between 10-21 h. Data obtained from a second experiment conducted to examine the effects of inhibin and follistatin on anterior pituitary gonadotropin responses to LHRH were consistent with in vitro data showing direct pituitary effects of the gonadal polypeptides. Collectively, these results demonstrate that both purified porcine follistatin and recombinant human inhibin A profoundly suppress serum FSH levels in a dose- and time-dependent manner, with inhibin being more potent in this regard. Whereas the onset of action is similar for the two polypeptides, the duration of action of follistatin is longer than that for inhibin, suggesting, among other factors, different metabolic clearance rates or different pretranscriptional mechanisms of action of follistatin and inhibin.

Animals↗

PubMed1991

Follistatin binds to both activin and inhibin through the common subunit.

Inhibin, activin, and follistatin are three families of polypeptides originally isolated and characterized from ovarian follicular fluid based on their modulation of FSH release from pituitary cell culture. In addition to their effects on FSH synthesis and secretion, inhibin and activin have other biological functions. By contrast, the physiological significance of follistatin was obscure, until it was discovered that follistatin is a binding protein to activin. Since activin binds to follistatin, it is imperative to determine the nature of the activin/follistatin binding complex. Moreover, because inhibin contains a beta-subunit derived from activin, it is important to determine whether inhibin will also bind follistatin. Using a double-ligand blotting technique, we have determined that activin-A has two binding sites for follistatin, whereas inhibin-A has only one binding site for follistatin. Therefore, these results suggest that follistatin binds to both activin and inhibin through the common beta-subunit.

Activins↗

PubMed1991

Characterization of an insulin-like growth factor binding protein (IGFBP-4) produced by the B104 rat neuronal cell line: chemical and biological properties and differential synthesis by sublines.

A previous report from our laboratory described an approximately 30 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) that inhibited the binding of insulin-like growth factor I (IGF-I) by its receptor and was secreted by a subline of the B104 rat neuronal cell line. To better understand the biology of this IGFBP, it was purified from media conditioned by these B104 cells, and the chemical and biological properties of the protein were examined. The IGFBP existed as a 24 kDa form and a 28 kDa form when the conditioned media were analyzed by ligand blot. Deglycosylation studies indicated the 28 kDa species was the N-linked glycosylated form of the 24 kDa IGFBP. Multiple forms at both mol wts were found using two-dimensional electrophoresis, suggesting that there were posttranslational modifications in addition to glycosylation. The amino acid sequence of the 12 amino-terminal residues was identical to that of rat IGFBP-4. Increased synthesis of IGFBP-4 by the subline contrasted with neglible production by other B104 cells. Blot hybridization with rat IGFBP-4 complementary DNA showed differential expression of a 2.6 kilobase transcript among B104 cell lines that correlated with quantities of IGFBP-4 secreted in media. The difference persisted even when the cells were xenografted into athymic nude mice. Purified IGFBP-4 inhibited the binding of [125I]IGF-I by its receptor and blunted stimulation of [3H]thymidine incorporation by IGF-I. These findings suggest a role for IGFBP-4 in neural cell function and indicate the B104 cell lines may be a useful model for further examination of IGFBP-4 biology.

Amino Acid Sequence↗

PubMed1991

Cyclic changes in follistatin messenger ribonucleic acid and its protein in the rat ovary during the estrous cycle.

The purpose of this research was to characterize the localization of follistatin mRNA and protein in the adult rat ovary during the 4-day estrous cycle. Analysis of ovarian sections using in situ hybridization and immunohistochemistry demonstrated the presence of follistatin messenger RNA (mRNA) and its protein in granulosa and luteal cells; no follistatin (message or protein) was detected in any of the other ovarian cell types. An important observation was that the intensity of follistatin signals changed during granulosa differentiation and the estrous cycle. During folliculogenesis, the first detectable hybridization signal appeared in the granulosa cells of secondary follicles, but the signal was weak. However, when a preantral follicle reached the early tertiary stage (beginning antrum formation), the message signal was very strong, being expressed in all granulosa cells of all such follicles (300-400 microns in diameter). In atretic follicles, follistatin mRNA was localized to granulosa cells, but only during the early stages. The above hybridization pattern of follistatin mRNA in prenatral and atretic follicles appeared constant throughout the estrous cycle. Interestingly, immunohistochemistry studies showed that the follistatin protein was detected only in certain follicles, being restricted to those which were healthy. On the morning of estrus, the follistatin protein was localized to a subpopulation of early tertiary follicles, presumably the dominant follicles selected to ovulate in the next cycle. As the dominant preovulatory follicles matured through diestrus and proestrus, the follistatin mRNA and protein signals appeared more intense in the granulosa cells. After ovulation, the hybridization and immunohistochemical signals continued to be strong in the newly formed corpora lutea on estrus morning. After luteolysis on diestrus-I, neither the follistatin message nor the protein was detectable in the corpora lutea. In conclusion, these results suggest that the follistatin message is present in all the granulosa cells of every developing follicle throughout the estrous cycle, but the follistatin protein appears to be present in only the selected dominant follicles. Accordingly, the possibility that follistatin might be an important regulatory molecule for selection/atresia should be considered.

Animals↗