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Biomedical subjects

N Ling

Publications and source records attributed to N Ling.

At least 127 records · Page 7Linked to original sources
PubMed1991

Recombinant expression of human follistatin with 315 and 288 amino acids: chemical and biological comparison with native porcine follistatin.

Follistatin is a glycosylated monomeric protein originally isolated from ovarian follicular fluid based on its ability to specifically inhibit pituitary FSH release. To further explore the physiological role of follistatin, we have expressed recombinant human follistatins with 315 (rhFS-315) and 288 (rhFS-288) amino acids in Chinese hamster ovary cells under the control of the simian virus-40 promoter. The two types of FS originated from alternatively spliced mRNAs and rhFS-315 differed from rhFS-288 by having an extra 27-amino acid sequence at the carboxyl-terminal. The yield of the purified rhFS-315 and rhFS-288 after a single step of affinity chromatography on an activin-coupled Affi-Gel column was 3-5 mg/liter conditioned medium. Using the rhFS-315 and rhFS-288 as molecular mass markers, Western blotting with FS carboxyl-terminal-specific antibodies demonstrated that the majority of native FS isolated from porcine ovarian follicular fluid was neither FS-315 nor FS-288, but was composed of 300 amino acids in various forms of glycosylation. This finding is consistent with our earlier results obtained from tryptic peptide fragment analysis of native FS. Only a very small percentage (less than 1%) of native porcine FS was FS-288. In cultures of rat anterior pituitary cells, rhFS-315 (ED50, 115.2 +/- 16.2 pM) is equipotent to native porcine FS (ED50, 86.7 +/- 14.1 pM) on the suppression of FSH release, but, surprisingly, rhFS-288 (ED50, 9.6 +/- 2.2 pM) is 8-10 times more potent than the native protein, similar to the potency of inhibin-A (ED50, 8.6 +/- 0.9 pM). Interestingly, when the in vivo FSH-suppressing activity of rhFS-288 was compared to that of inhibin-A in 1-week ovariectomized adult rats, it was found that rhFS-288 was more potent and longer acting than inhibin-A. Hence, these results indicate that FS-288 is probably one of the most potent natural FSH suppressors.

Amino Acid Sequence↗
PubMed1991

Tissue-specific expression of four insulin-like growth factor-binding proteins (1, 2, 3, and 4) in the rat ovary.

We investigated the possible presence of the mRNAs encoding four insulin-like growth factor-binding proteins (IGFBP-1, -2, -3, and -4) in the rat ovary. It has been demonstrated previously by Northern analysis that adult rat ovaries contain mRNA transcripts for IGFBP-2 and -3. Here we show by Northern analysis that adult rat ovaries contain mRNA for IGFBP-4, but the mRNA for IGFBP-1 was below the limit of detection. Using in situ hybridization, IGFBP-1 mRNA was not detected in any of the ovaries tested. The IGFBP-2 mRNA was localized specifically in thecal interstitial cells (TIC) and secondary interstitial cells of all ovaries. The IGFBP-2 signal was very strong in TIC of all Graafian follicles (healthy and atretic) and very strong in all secondary interstitial cells located in different regions of the ovary, but weak in TIC of preantral follicles. The IGFBP-3 hybridization signal was localized specifically to some corpora lutea. Here the hybridization single for IGFBP-3 was strong and distributed throughout the corpus luteum, strong but localized to only some luteal cells, or not detectable. The IGFBP-4 mRNA was localized almost exclusively to granulosa cells of atretic follicles. The intensity of the IGFBP-4 signals appeared to increase as the level of atresia increased. In some cases, IGFBP-4 signals were detected in the TIC, but they were weak and variable. These results show that the mRNAs for IGFBP-2, -3, and -4 are localized to the interstitial cells, corpora lutea, and atretic granulosa cells, respectively. The tissue-specific synthesis of IGFBP subtypes in specialized ovarian cells provides an excellent system to study the manner in which IGFBP synthesis is controlled and their potential role as an autocrine and paracrine factors.

Animals↗
PubMed1991

Isolation and molecular cloning of insulin-like growth factor-binding protein-6.

Insulin-like growth factors (IGFs) together with their binding proteins (BPs) are potential regulators of folliculogenesis in mammalian ovary. To identify the various species of IGFBPs present in the ovary, we have undertaken a comprehensive purification scheme using gel filtration, ligand-affinity chromatography, and several steps of reverse phase HPLC to isolate all of the BPs in pig ovarian follicular fluid. Our effort yielded five distinct IGFBPs, and upon analysis, they were found to correspond to the previously identified human and rat IGFBP-2, -3, -4, -5, and -6. IGFBP-1 was not found in the pig ovarian follicular fluid under our experimental procedure. Of the six known classes of IGFBPs, the complete primary structures of the first five have been determined, but not IGFBP-6. Using amino acid sequence information from a tryptic fragment of pig IGFBP-6 to prepare a probe, cDNA clones encoding rat and human IGFBP-6 have been isolated and characterized. The deduced amino acid sequence revealed that rat IGFBP-6 contains 201 amino acids with a calculated mol wt of 21,461, while the human homolog contains 216 amino acids with a calculated mol wt of 22,847. In addition, a distinctive feature of human and rat IGFBP-6 is that they lack, respectively, two and four of the 18 homologous cysteines that are present in all other five IGFBPs. The missing cysteines in IGFBP-6 resulted in the absence of the invariant Gly-Cys-Gly-Cys-Cys sequence in the amino-terminal region of the molecule. Human IGFBP-6 possesses a single Asn-linked glycosylation site near the carboxyl-terminal, whereas no potential Asn-linked glycosylation sites are present in the rat sequence. A single 1.3-kilobase IGFBP-6 mRNA was detected by Northern analysis in all rat tissues examined, including testis, intestine, adrenal, kidney, stomach, spleen, heart, lung, brain, and liver, indicating that this BP is a ubiquitous protein. The chromosome location of the IGFBP-6 gene in human has been determined using polymerase chain reaction on somatic cell hybrid DNAs of human and hamster, and the results showed that it is located on chromosome 12.

Amino Acid Sequence↗
PubMed1991

Growth factor-mediated regulation of aromatase activity in human skin fibroblasts.

We investigated the effects of various hormones and growth factors on aromatase activity in cultured human skin fibroblasts. Several potential trophic factors were tested for their ability to modify basal aromatase activity or the response to dibutyryladenosine 3',5'-cyclic monophosphate and dexamethasone because (i) no endogenous ligand has been identified that is responsible for stimulating aromatase activity in the periphery, and (ii) dexamethasone and cAMP analogs can increase this enzyme's activity in fibroblasts. The effect of insulin and insulin-like growth factors were examined in closer detail because of the clinical association between insulin and hyperandrogenism. Pituitary hormones and hypothalamic releasing factors, such as human ACTH (10 nM), beta-endorphin (10 nM), beta-lipotropin (10 nM), alpha-MSH (10 nM), gamma 3-MSH (10 nM), ovine luteinizing hormone (10 ng/ml), ovine follicle-stimulating hormone (10 ng/ml), ovine thyroid-stimulating hormone (10 ng/ml), rat growth hormone (10 ng/ml), rat prolactin (10 ng/ml), rat corticotropin-releasing factor (10 nM), luteinizing hormone-releasing factor (10 nM), thyrotropin-releasing factor (10 nM), human growth hormone-releasing factor (10 nM), and somatostatin (10 nM), have no significant effects on aromatase activity. Porcine inhibin A (10 ng/ml) and porcine activin AB (10 ng/ml), two ovarian hormones with structural transforming homology to transforming growth factor-beta, also have no effect on aromatase activity. Although basic fibroblast growth factor (1-100 ng/ml), acidic fibroblast growth factor (1 ng/ml), epidermal growth factor (1 ng/ml), platelet-derived growth factor (1 ng/ml), tumor necrosis factor (1 ng/ml), and transforming growth factor-beta 1 (1 ng/ml) have no effect on basal aromatase activity in human skin fibroblasts, all of these growth factors inhibited the ability of dibutyryladenosine 3',5'-cyclic monophosphate to stimulate aromatase activity. In contrast, both insulin (100 pg/ml-10 ng/ml) and insulin-like growth factor-1 (1-100 ng/ml) had no effect on cAMP-stimulated aromatase but potentiated the action of dexamethasone (100 nM). Thus, there is a clear distinction between the effects of dexamethasone and cAMP on peripheral aromatase. On the basis of the results presented here, it is interesting to speculate that the hyperandrogenism that is often associated with insulin resistance may be due to a combination of growth factor-mediated inhibition of aromatase activity and the failure of peripheral tissues to respond to insulin and metabolize androgens to estrogens.

Activins↗
PubMed1991

Radioimmunoassay of inhibin based on synthetic human inhibin alpha-chain peptide.

Polyclonal rabbit antisera were produced against cyclic human inhibin [(Cys6, Tyr7) alpha-(6-30)NH2] peptide, covalently conjugated to bovine serum albumin. The tyrosine residue introduced at position 7 facilitated the oxidative incorporation of radiolabel (125I) to yield a tracer with specific activity of 73.9 Ci/g. These reagents were used to develop a homologous equilibrium radioimmunoassay for human inhibin, with polyethylene glycol, 200 g/L, serving as the separation phase. At a detection limit of 2 micrograms/L (n = 7), immunoactive inhibin was detectable in human pre-ovulatory follicular fluid (128 micrograms/L), seminal plasma (2374 micrograms/L), amniotic fluid (66 micrograms/L), and placental extract (347 micrograms/L). We also demonstrated inhibin immunoreactivity in biological fluids from other mammalian species: macaque, chimpanzee, porcine, and bovine, but not rodent (guinea pig). Although the antisera were raised against a nonbioactive inhibin peptide, immunoglobulins fractionated on Protein A-Sepharose neutralized the bioactivity of human ovarian inhibin. Further characterization of inhibin immuno- and bioactivity was undertaken with immobilized heparin, divalent metal cations, and dye ligands. Only heparin-Sepharose distinguished between immuno- and bioactive inhibin.

Amniotic Fluid↗
PubMed1990

Insulin-like growth factor binding protein measurement: sodium dodecyl sulfate-stable complexes with insulin-like growth factor in serum prevent accurate assessment of total binding protein content by ligand blotting.

The possibility that sodium dodecyl sulfate (SDS)-stable complexes of insulin-like growth factor I (IGF-I) and its binding proteins (IGF-BP) exist in rat serum has been examined by using SDS-polyacrylamide gel electrophoresis (PAGE) followed by both [125I]IGF-I ligand blotting and immunoblotting with antisera directed against either IGF-BP3 or IGF-I. While ligand blotting of rat serum only revealed free IGF-BP subunits (Mr approximately 50, 35, and 30 kDa), immunoblotting with either the IGF-BP3 antiserum or IGF-I antiserum revealed major immunoreactive bands with higher molecular weights (greater than 110, approximately 100, and approximately 84 kDa). The IGF-BP3 antiserum also stained the 50-kDa form of the serum IGF-BP. Specifically stained protein bands were identified by comparison with control immunoblots incubated with normal rabbit serum. Treating the serum with 0.1 N HCl prior to electrophoresis reduced the amount of high molecular weight IGF-BP3 immunoreactive species, with a concomitant increase in the amount of the 50-kDa form. A similar result was obtained if the samples were boiled prior to electrophoresis. These data indicate that not all IGF-BP/IGF complexes may dissociate under normal SDS-PAGE conditions. Therefore, data obtained by using ligand blotting alone may underestimate the amount of total IGF-BP present, especially if the mixture being analyzed also contains large amounts of IGF.

Animals↗
PubMed1990

Insulin-induced hypoglycemia, L-dopa and arginine stimulate GH secretion through different mechanisms in man.

We sought to clarify the mechanisms of growth hormone (GH) secretion induced by insulin hypoglycemia, L-dopa, and arginine in man. The secretion of GH as measured by increased plasma level, in response to oral administration of 500 mg L-dopa or 30 min-infusion of arginine, was not modified by prior intravenous administration of 200 micrograms GH-releasing hormone (GHRH). It was, however, completely blocked by preadministered 50 micrograms SMS201-995, a long-acting somatostatin (SRIH) analog. GH release with 200 micrograms GHRH was completely blocked by 100 micrograms SMS201-995. GH secretion caused by insulin-induced hypoglycemia was significantly reduced but still present after administration of 100 micrograms of the analog. These results suggest that a suppression of SRIH release may be partially involved in the stimulatory mechanism of GH secretion by L-dopa. Coadministration of GHRH accentuated the stimulatory effect of arginine on GH secretion. Arginine significantly raised plasma TSH levels. These findings suggest that arginine suppresses SRIH release from the hypothalamus to cause GH secretion because SRIH suppresses TSH secretion. It is also suggested that some factor (or factors) other than GHRH and SRIH are involved in the mechanism by which insulin-induced hypoglycemia stimulates GH secretion, because the effect of insulin was not fully blocked in the presence of SRIH analog. Thus all the tests for GH release appear to act via different mechanisms.

Adult↗
PubMed1990

Determinants of plasma glucose level and diabetic status in a northern Canadian Indian population.

We conducted a cross-sectional survey of 704 Indians aged 20 to 64 years in six remote communities in northern Ontario and Manitoba to determine the factors associated with the fasting plasma glucose and glycosylated hemoglobin levels and diabetic status, defined by past history and current fasting plasma glucose level. Multivariate analyses for the 671 subjects with complete data showed that triglyceride level, age and body mass index (BMI) were significant predictors of the log fasting plasma glucose level and the log glycosylated hemoglobin level; for the latter, waist/hip ratio, history of diabetes mellitus among first-degree relatives and low level of education were additional predictors. Significant risk factors for diabetes as a dichotomous variable included triglyceride level, age, BMI and family history of diabetes. Although energy intake per unit of body weight was lower among subjects with diabetes than those without diabetes, possibly reflecting the lower physical activity level of diabetic subjects, the former consumed significantly more "calorie-adjusted" protein and less carbohydrate than the latter. The findings are consistent with studies in other populations. Further study is needed to determine the natural history of diabetes and its metabolic consequences and to assess the effect of dietary alteration and promotion of physical activity on the incidence of the disease.

Adult↗
PubMed1990

Synthetic analogs of growth hormone-releasing factor with antagonistic activity in vitro.

Analogs of human and rat growth hormone-releasing factor (hGRF and rGRF), related to [D-Arg2]hGRF(1-29)NH2, were synthesized by solid phase methodology. Their capacity to inhibit growth hormone secretion stimulated by hGRF(1-44)NH2 was tested on rat anterior pituitary cells in monolayer culture. Among the analogs of hGRF, [D-Arg2,29,Arg30]hGRF(1-30)NH2 showed the highest antagonistic potency of 3.64 relative to [D-Arg2]hGRF(1-29)NH2 = 1. However, the most potent analog synthesized thus far was [N-Ac-His1,D-Arg2,Ala15]rGRF(1-29)NH2, which showed a relative potency of 27.7.

Amino Acid Sequence↗
PubMed1990

Structural characterization of a follicle-stimulating hormone action inhibitor in porcine ovarian follicular fluid. Its identification as the insulin-like growth factor-binding protein.

Recently, an inhibitory polypeptide that could block the follicle-stimulating hormone-induced estradiol and progesterone production in rat ovary granulosa cells has been isolated from porcine ovarian follicular fluid. Amino-terminal sequence analysis of the purified inhibitor suggests that it could be the porcine congener of the 53-kDa subunit of the growth hormone-dependent insulin-like growth factor binding protein (IGF-BP3). Using amino acid sequence information derived from the purified inhibitor to construct oligonucleotide probes, we have now identified the complementary deoxyribonucleic acids (cDNAs) encoding the inhibitory polypeptide from a porcine liver and a porcine ovary library. The nucleotide and predicted amino acid sequences revealed that the cDNAs indeed encode the porcine homolog of the recently characterized human IGF-BP3. The mature polypeptide consists of 266 amino acids, which is 2 amino acids longer than the human sequence. Between the two species, there are 42 amino acid substitutions, but the 18 cysteines and the three Asn-linked glycosylation sites are totally conserved. A single mRNA species of 2.6 kilobases encoding the IGF-BP3 was detected in porcine gonadal, brain, and liver tissues by Northern analysis.

Amino Acid Sequence↗
PubMed1990

Atrial natriuretic factor increases cyclic GMP and cyclic AMP levels in a directly photosensitive pineal organ.

Atrial natriuretic factor (ANF) stimulates accumulation of cyclic GMP in a photosensitive organ, as evidenced for the first time in cultured trout pineals. Stimulation was rapid (within a few min), dose-dependent, and stronger in organs cultured in darkness than in those cultured under light. After 30 min in the dark, (i) cyclic AMP levels were slightly increased at 10(-7) mole/l of ANF, (ii) cyclic GMP and cyclic AMP increased dramatically after inhibition of the phosphodiesterases by isobutylmethylxanthine (IBMX), (iii) ANF and IBMX effects were more than additive on cyclic GMP, (iv) pertussis toxin decreased the cyclic GMP response to ANF. These responses were affected by light. The possibility that cyclic GMP might be a second messenger of both light and chemical (ANF) inputs, in pineal photoreceptor cells, is hypothetized.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Immunocytochemical demonstration of GAP-like immunoreactive neuronal elements in the human hypothalamus and pituitary.

GnRH-associated peptide (GAP)-like immunonreactive elements located in the human hypothalamus were investigated by PAP immunocytochemistry using specific antiserum against [pro-GnRH (14-69) OH]. Immunoreactive neuronal perikarya were distributed in the MPOA, PVN and infundibular nucleus, with the largest numbers of GAP-like immunoreactive perikarya found in the infundibular nucleus. We also detected the coexistence of GAP-like and GnRH-like immunoreactivities in the same neuronal perikarya in the MPOA by using a double immunolabelling procedure. In addition to the above regions immunoreactive neuronal perikarya were present in the region dorsal to the medial mammillary nucleus. GAP-like immunoreactive fibers were distributed in same areas that immunoreactive perikarya were observed. Many immunoreactive terminals were found adjacent to capillaries in the infundibulum. Immunoreactive dots, presumably terminals, were observed in the posterior pituitary and these were particularly evident along the margin adjacent to the anterior pituitary. The distribution pattern and density of GAP-like immunoreactive neuronal elements are compared with those of other mammalian species. We also compared GAP-like immunoreactive elements with that of GnRH as has been previously observed in the human hypothalamus.

Adult↗
PubMed1990

Brain corticotropin-releasing factor acts as inhibitor of stress-induced gastric erosion in rats.

Gastric lesions are known to be caused by stress. Corticotropin-releasing factor (CRF) is a key peptide initiating various stress response. This study was designed to investigate how brain CRF is involved in the occurrence of stress-induced gastric erosion in rats. Intracerebroventricular (icv) administration of CRF suppressed the occurrence of gastric erosion induced by water-immersion restraint stress, and its suppressive effect was blocked by coadministration of a CRF receptor antagonist in rats. The peripheral administration of CRF had no influence on the occurrence of erosion. The icv administration of a CRF receptor antagonist or anti-rat CRF gamma-globulin increased gastric erosion induced by the stress. Ganglionic blockade with chlorisondamine, muscarinic blockade with atropine, or bilateral adrenalectomy by itself significantly inhibited the occurrence of stress-induced gastric erosion, and no additional effect of CRF on these treatments-induced inhibition of erosion was found. These results, therefore, suggest that the occurrence of stress-induced gastric erosion is mediated by the autonomic nervous system- and adrenal-dependent pathway, and that brain CRF reduces the occurrence of stress-induced gastric lesions by acting on its specific receptor within the central nervous system, probably through the autonomic nervous system- and adrenal-dependent mechanism.

Animals↗
PubMed1990

Processing of prosomatostatin.

Several peptides are generated from prosomatostatin (proSS) in addition to somatostatin-14 (SS-14) and somatostatin-28 (SS-28). These are SS-28(1-12), proSS(1-76) and, as shown more recently, proSS(1-63) and antrin. Important variations in the proportion of these molecular forms are seen among different tissues and among different species. Processing of the precursor in the human brain yields minimal quantities of SS-28(1-12) and high levels of proSS(1-76), namely in cortical areas and in the bed nucleus of the stria terminalis. This is in contrast with findings in rat brain, where SS-28(1-12) is a predominant molecular form. Antrin, which corresponds to proSS(1-10), reaches its highest concentration in the antral portion of the stomach (117 +/- 13 pmol/g wet weight), where it is found in secretory granules of delta cells. We observed an inverse relationship between levels of antrin and proSS(1-63) after chromatography of various tissue extracts. This suggests a precursor-product relationship between these two peptides.

Amino Acid Sequence↗
PubMed1990

Novel ovarian regulatory peptides: inhibin, activin, and follistatin.

Based on the extensive amount of research on inhibin and related polypeptides accomplished during the past 5 years, the inhibin concept put forth more than 50 years ago has not only become well established but also more complex than originally imagined. The closed-loop feedback mechanism of ovarian inhibin and pituitary FSH has been joined by possible "inhibin-like" actions of follistatin and FSH-stimulatory effects of activin. In addition, in vitro experiments suggest possible autocrine and paracrine functions for the gonadal polypeptide hormones. Figure 3 shows a simplistic diagram summarizing our current understanding of inhibin/activin and follistatin action along the hypothalamic-pituitary-gonadal axis. Hopefully, research in the coming years will allow us to remove the many question marks still remaining but will undoubtedly add others.

Activins↗