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Results for “Bioactivation”

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PubMed2004

Circulating tumour necrosis factor-alpha bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response.

Our objective was to clarify the heterogeneity in response to infliximab treatment in rheumatoid arthritis (RA); to this end, a bioassay was designed to explore the contribution of circulating tumour necrosis factor (TNF)-alpha bioactivity and its possible link to response. The bioassay is based on the induction of IL-6 and osteoprotegerin (OPG) production by synoviocytes in response to TNF-alpha. RA synoviocytes were cultured with TNF-alpha (5 ng/ml) and 42 RA plasma samples collected just before starting therapy. Levels of IL-6 and OPG were measured in supernatants. In 20 of the patients, plasma samples collected before and 4 hours after the first and the ninth infusions were tested in the same way. Plasma concentrations of TNF-alpha and p55 and p75 soluble receptors were measured using ELISA. TNF-alpha induced IL-6 and OPG production by synoviocytes, which was further increased with patient plasma dilutions and inhibited by infliximab. With plasma samples obtained before the first infusion, the IL-6-induced production was greater in patients with a good clinical response than in the poor responders (44.4 +/- 23.3 ng/ml versus 27.4 +/- 20.9 ng/ml; P = 0.05). This high circulating TNF-alpha bioactivity was strongly inhibited with the first infliximab infusion. The difference between IL-6 levels induced with plasma samples obtained before and 4 hours after the first infusion was greater in patients with a good clinical response (40.0 +/- 23.7 ng/ml versus 3.4 +/- 10.0 ng/ml; P = 0.001). Similar findings were obtained for OPG production (7.0 +/- 6.2 ng/ml versus 0.0 +/- 3.0 ng/ml; P < 0.05). Levels of circulating TNF-alpha bioactivity were predictive of clinical response to TNF-alpha inhibition, confirming a key role for TNF-alpha in these RA patients.

Adult↗

PubMed2005

Molecular and mechanistic characterization of platelet-activating factor-like bioactivity produced upon LDL oxidation.

Oxidation of LDL is thought to be involved in both initiating and sustaining atherogenesis through the formation of proinflammatory lipids and the covalent modification of LDL particles. Platelet-activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator involved in inflammation. Upon oxidation of LDL, oxidized phospholipids with PAF-like structure are generated, and some of them may act via the PAF receptor. We evaluated the contribution of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and of other PAF analogs on the PAF-like bioactivity formed upon Cu2+-initiated oxidation of LDL. Reverse-phase HPLC purification and electrospray ionization-MS analyses showed that upon oxidation of LDL with inactivated PAF-acetylhydrolase (PAF-AH), C16:0 PAF accounted for >30% of PAF-like biological activity and its sn-2 butenoyl analog accounted for >50%. However, upon LDL oxidation in the presence of exogenous 1-0-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) without PAF-AH inactivation, C16:0 PAF formation accounted for >90% of the biological activity recovered. We suggest that the C16:0 PAF, despite being a minor constituent of the LDL peroxidation products, may contribute substantially to the bioactivity formed in oxidized LDL. The higher bioactivity of C16:0 PAF, and the higher selectivity of the LDL-attached lyso-PAF transacetylase toward very short acyl chains [acetate (C2) vs. butanate (C4)], may explain the contribution described above.

Chromatography, High Pressure Liquid↗

PubMed2005

The development of bioactive triple helix-forming oligonucleotides.

We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in mammalian cells. We have described psoralen-conjugated TFOs containing 2'-O-methyl (2'OMe) and 2'-O-aminoethoxy (AE) ribose substitutions. TFOs with a cluster of 3-4 AE residues, with all other sugars as 2'OMe, were bioactive in a gene knockout assay in mammalian cells. In contrast, TFOs with one or two clustered, or three dispersed, AE residues were inactive. Thermal stability analysis of the triplexes indicated that there were only incremental differences between the active and inactive TFOs. However the active and inactive TFOs could be distinguished by their association kinetics. The bioactive TFOs showed markedly greater on-rates than the inactive TFOs. It appears that the on-rate is a better predictor of TFO bioactivity than thermal stability. Our data are consistent with a model in which a cluster of 3-4 AE residues stabilizes the nucleation event that precedes formation of a complete triplex. It is likely that triplexes in cells are much less stable than triplexes in vitro probably as a result of elution by chromatin-associated translocases and helicases. Consequently the biologic assay will favor TFOs that can bind and rebind genomic targets quickly.

Animals↗

PubMed1998

Soluble tumor necrosis factor-alpha (TNF-alpha) receptors in human colostrum and milk bind to TNF-alpha and neutralize TNF-alpha bioactivity.

We used column chromatography, affinity binding, and bioassay methods to address whether the soluble tumor necrosis factor (TNF)-alpha receptors present in human colostrum and milk bind to and modify TNF-alpha bioactivity. In gel chromatography experiments, soluble TNF-alpha receptor I (sTNFRI) and sTNFRII in human colostrum sequentially increased their molecular sizes from 49 kD to 71 kD and 60 kD, respectively, after addition of increasing molar excesses of recombinant TNF-alpha. Application of colostrum to a TNF-alpha affinity matrix followed by washing and elution resulted in 2925-fold enrichment of sTNFRI, consistent with sTNFRI binding to the TNF-alpha affinity matrix. In other samples of colostrum and milk, the content of both sTNFRI and sTNFRII decreased significantly after passage over the matrix, but the material eluted from the matrix lost the ability to rebind to the TNF-alpha and was not active in a WEHI-13var bioassay for TNF-alpha. Specimens of human colostrum and milk diluted 1:16 shifted the LD50 for TNF-alpha 4-fold in this bioassay, and milk protection of WEHI-13var cells against TNF-alpha was significantly diminished after passage down the TNF-alpha affinity matrix (p < 0.001). Affinity purification of milk sTNFRI using polyclonal anti-sTNFRI produced fractions containing proteins of 30 kD, which could be visualized by Western blot using polyclonal anti-sTNFRI. Addition of this fraction to the WEHI-13var bioassay reversed the effects of 10 pg/mL TNF-alpha in the assay. These data demonstrate that sTNFRI and II from human colostrum and milk bind to TNF-alpha, that both colostrum and milk interfere with the bioactivity of TNF-alpha, and that affinity-purified sTNFRI from human milk blocks the bioactivity of TNF-alpha. These effects may contribute to the anti-inflammatory character of human colostrum and milk.

Animals↗

PubMed1980

Modulation of serum and pituitary luteinizing hormone bioactivity by androgen in the rat.

The biological and immunological activities of serum and pituitary LH were analyzed in normal and castrated rats using the rat interstitial cell testosterone assay and RIAs for intact rat LH (rLH), rLH alpha, and rLH beta. After castration, the bioactivity to immunoactivity ratio of both serum and pituitary LH decreased by about 50% between the 5th and 25th days and returned to normal by 60 days. This decrease in the bioactivity to immunoreactivity ratio of LH in castrated males was not due to increased subunit production and was prevented by the administration of testosterone propionate (50 micrograms/day) from the time of castration. These observations indicate that androgens of their metabolites modify the bioactivity of the circulating and stored forms of the LH molecule.

Animals↗

PubMed1984

Changes in inhibin-like bioactivity in ovulatory and atretic follicles and utero-ovarian venous blood after prostaglandin-induced luteolysis in heifers.

The objectives of this study were to develop a bioassay for measuring inhibin bioactivity in untreated samples of bovine follicular fluid (BFF) and then examine changes in inhibin bioactivity in ovulatory and atretic follicles and utero-ovarian venous blood during the periovulatory period in heifers. A rat pituitary cell culture system was used to bioassay inhibin-like activity. Addition of 0.005 to 1 microliter untreated (whole), unfiltered charcoal-stripped, or filtered whole BFF to pituitary cultures caused a linear suppression of LHRH-induced FSH release but had no effect on LH secretion. Steroids in BFF did not suppress FSH secretion, since removal of steroids from BFF with charcoal did not remove the FSH-suppressive activity in BFF. Addition of ether extracts of BFF caused a slight but nonparallel suppression of FSH secretion; however, heating these extracts removed most of this suppressive activity. Removal of BFF from pituitary cultures completely restored the capacity of pituitary cultures to respond to LHRH. It was concluded that the inhibin bioassay was specific for detecting inhibin-like activity in fluids from individual follicles without interference of steroids. Within 12 h after a prostaglandin (PG) injection during the luteal phase of heifers, LH levels in serum increased 2- to 4-fold and remained at this level until the occurrence of the preovulatory gonadotropin surge. In contrast, FSH did not change before the gonadotropin surge. Inhibin bioactivity was measured in all follicles (greater than or equal to 6 mm) 0, 12, 24, 36, 48, 60, and 72 h after and in utero-ovarian venous serum 0, 24, and 36 h after PG-induced luteolysis. From 0-36 h after PG administration, inhibin-like activity increased linearly in presumed ovulatory follicles and utero-ovarian venous serum. Then, from 48-72 h after PG treatment, before the preovulatory LH surge, inhibin activity decreased in ovulatory follicles. After the surge but before ovulation, inhibin-like activity increased in ovulatory follicles. Inhibin-like activity in atretic follicles did not change after PG treatment and was lower in atretic than ovulatory follicles. Since a single hypothalamic releasing factor, LHRH, may control the secretion of LH and FSH, increased secretion of inhibin from preovulatory follicles before the preovulatory LH and FSH surges could account for the absence of a presurge rise in FSH in blood, as was observed for LH during this time in heifers. Diminished follicular production of inhibin during the gonadotropin surge could explain the preovulatory release of FSH along with LH during this time.

Animals↗

PubMed1985

Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides.

A soluble metalloendopeptidase identified in rat brain, preferentially cleaves bonds in peptides having hydrophobic amino acid residues in the P1, P2, and P3' positions. (The nomenclature of T. Schechter and A. Berger is used to describe the interaction between enzyme and substrate. The amino acid residues in the substrate are designated as P1, P2, P3 etc. in the N-terminal direction and P1', P2', P3' etc. in the C-terminal direction from the bond undergoing cleavage. The corresponding subsites in the enzyme are identified by the letter S.) The degradation of a series of biologically active peptides and their affinity toward the enzyme was studied. Dynorphin-(1-8), alpha-neo-endorphin, and beta-neo-endorphin are rapidly hydrolyzed to form leu-enkephalin, whereas bovine adrenal medulla dodecapeptide is hydrolyzed to form met-enkephalin. The enzyme, however, does not cleave a larger precursor molecule of metenkephalin, such as bovine adrenal medulla docosapeptide. Several other bioactive peptides are also cleaved at sites consistent with our previously reported specificity studies. Met- and leu-enkephalin are resistant to hydrolysis. The binding affinity [as expressed by inhibitory constant (Ki) or Michaelis-Menten constant (Km) values] of several bioactive peptides such as dynorphin-(1-8), beta-neo-endorphin, neurotensin, angiotensin I, and bradykinin was found to be in the micromolar range. These peptides were also rapidly hydrolyzed by the enzyme, showing, as a result, high specificity constants (kcat/Km values). The highest enzyme activity was found in brain, testis, and in the anterior and posterior lobes of the pituitary, while the activity in such tissues as spleen, liver, kidney, lung, adrenals, and thyroid amounted to only 10-20% of that found in brain. This distribution of enzyme activity, together with its preference for oligopeptides as substrates, its ability to generate leu- and met-enkephalin from several larger peptide precursors, and its affinity toward several other bioactive peptides, suggests that the enzyme functions in the metabolism of neuropeptides.

Animals↗

PubMed1989

Bioactive follicle-stimulating hormone release in nutritionally growth-retarded ovariectomized lambs: regulation by nutritional repletion.

The nutritionally growth-retarded ovariectomized lamb provides a model system to investigate the regulation of immunoreactive and bioactive FSH release in the absence of feedback effects of ovarian products. We examined the acute effects of nutritional repletion on FSH release in this model. Five March-born lambs were weaned at 7 weeks of age and placed on a limited diet. All were ovariectomized at 31 weeks. At 37 weeks, they were fed ad libitum for 14 days. Blood samples were collected at 12 min intervals for 4 h on day -1 (restricted) and days 2, 7, and 14 of ad libitum feeding, and serum FSH concentrations were measured by both RIA and a Sertoli cell aromatase in vitro bioassay. Mean concentrations of serum immunoreactive FSH (I-FSH) and the bioactive FSH (B-FSH) increased 2- and 3-fold, respectively, after ad libitum feeding for 14 days. Pulse analyses by two objective computerized programs, DETECT and CLUSTER, revealed the presence of FSH pulses (predominantly B-FSH) during the restricted phase, as well as ad libitum fed phases, at times where I-LH pulses were not detected. In contrast to the increases in I-LH pulse frequency during ad libitum fed states, FSH pulse frequency remained stable whereas pulse amplitude increased. In addition, the overall B-FSH pulse amplitudes were 57 +/- 7% (mean +/- SE of three different pulse analyses) greater than I-FSH pulse amplitudes, implying preferential enrichment of FSH bioactivity. The results suggest that the mechanisms governing both the quantitative and qualitative changes in circulating FSH concentrations are extremely sensitive to changes in level of nutrition. The asynchronous occurrence of FSH pulses in the absence of I-LH pulses suggests that FSH may be more sensitive to small changes in GnRH secretion than I-LH, or that a hypothalamic releasing factor specific for FSH may be released.

Animals↗

PubMed1992

Circulating bioactive follicle-stimulating hormone and less acidic follicle-stimulating hormone isoforms increase during experimental induction of puberty in the female lamb.

The pubertal process with its multifaceted neuroendocrine control provides an excellent model for the study of the regulation of FSH heterogeneity. We tested the hypothesis that during the pubertal transition in the female lamb 1) an increase in both pituitary and circulating bioactive FSH concentrations occur and 2) that the increase in bioactivity is associated with a change in the distribution pattern of both pituitary and circulating FSH isoforms. Pituitary and serum immunoreactive (I), and bioactive (B, Sertoli cell bioassay) FSH concentrations were measured in six prepubertal lambs (18 +/- 1 weeks, 29.9 +/- 2.8 kg body weight; mean +/- SE) and compared to those of six others (24.2 +/- 2.2 weeks of age, 41.4 +/- 2.5 kg body weight) during the pubertal transition period. Puberty was synchronized by pulsatile iv administration of GnRH (2 ng/kg every 2 h for 24 h and then at hourly intervals for the next 12 h) in a manner mimicking the I-LH pulse patterns observed during the natural transition to adulthood. Blood samples were collected at 12-min intervals for 4 h from both groups of lambs; for the pubertal group this included the final 32-36 h of GnRH administration. At the end of the study, a 25 ml volume of peripheral blood was collected from both prepubertal and pubertal females for the determination of serum FSH distribution patterns; the lambs were then euthanised, and pituitaries were removed for determination of pituitary hormone content and FSH isoform distribution patterns. In addition, the distribution pattern of I-FSH isoforms in the pituitary and serum from both groups of lambs were compared. The pubertal stages of all lambs were verified by measuring the size of follicles, the circulating concentrations of estradiol (E2) and inhibin, and the I-LH pulse patterns. Prepubertal lambs had low frequency I-LH pulses, small (2-3 mm) size ovarian follicles and low circulating concentrations of E2 (4.1 +/- 0.4 pg/ml) and inhibin (38.0 +/- 2.9 U/ml WHO). By contrast, all the pubertal lambs had hourly I-LH pulse frequency (induced with exogenous GnRH), a large (5-6 mm) follicle (in one lamb a 4-mm follicle), follicular phase levels of E2 (7.1 +/- 0.8 pg/ml), and higher concentrations of inhibin (53.2 +/- 3.1 U/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Differential expression of epidermal growth factor receptor (EGF-R) gene and regulation of EGF-R bioactivity by progesterone and estrogen in the adult mouse uterus.

The present study examined several aspects of epidermal growth factor receptor (EGF-R) in the mouse uterus during the peri-implantation period and after ovarian hormone treatment of adult ovariectomized mice. The cell-specific distribution, regulation of expression, and binding kinetics were assessed by immunohistochemistry, Northern blot analysis, and ligand binding assays, respectively. Affinity cross-linking studies ascertained the size of the EGF-R, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase activity and receptor autophosphorylation. In the intact uterus and separated cell types, EGF-R was detected in the stroma, deciduum, and myometrium, but not in the luminal or glandular epithelium. Uterine EGF-R mRNA transcript profiles showed some differences between pregnant and ovariectomized mice regardless of steroid hormone treatments. Two major [6.5- and 2.7-kilobase (kb)] and two less abundant (9.6- and 5.0-kb) transcripts were detected in pregnant uterine poly(A)+ RNA. Three additional transcripts (< 2.0 kb) were detected in decidual poly(A)+ RNA, and a larger transcript (8.0 kb) was detected in uterine poly(A)+ RNA isolated from ovariectomized mice. Scatchard analysis of EGF binding also revealed apparent differences in binding kinetics between pregnant and ovariectomized mice, although EGF was cross-linked to a 170-kilodalton protein under these conditions. Two classes (Kd, approximately 0.2 and approximately 2.0 nM) of binding sites were noted in pregnant mice, whereas a single class (Kd, approximately 1.0 nM) was found in ovariectomized mice. 17 beta-Estradiol (E2) caused a rapid transient upregulation of uterine EGF-R mRNA levels and increased the number of EGF-binding sites in ovariectomized mice, as did an injection of progesterone (P4). However, the bioactivity of EGF-R could not be detected in uteri of ovariectomized mice treated with oil or P4. E2 treatment was found to be essential for EGF-R bioactivity. Taken together, the results suggest that in the adult mouse uterus, EGF-R status is influenced by factors other than P4 and E2, the epithelium is not the direct target for the actions of EGF-related growth factors as thought previously, the mitogenic effects of these growth factors on epithelial cells in vivo are perhaps mediated by other uterine cell-types expressing EGF-R, and, lastly, these growth factors are not likely to be functional in the uterus in the absence of estrogen. The present observations are supportive of the concept of paracrine or juxtacrine interactions between EGF-related growth factor ligands of luminal epithelial origin and blastocyst EGF-R in the process of implantation.

Animals↗

PubMed1995

Bioactive and immunoreactive prolactin variants in human milk.

Human milk from mothers of term (T) and preterm (PT) infants was collected during early (days 2-7), mature (2-16 weeks), or late (> 16 weeks) lactation. PRL-like bioactivity (B) was measured by Nb2 cell proliferation, and PRL immunoreactivity (I) was determined by RIA. PRL activity is reported in PRL equivalents (1 PRL equivalent = 1 ng NIDDK reference material). Milk from early lactation contained significantly greater PRL-like B compared to I (T:B, 132.5 +/- 13.0; I, 83.43 +/- 12; PT:B, 195.8 +/- 56; I, 74.45 +/- 13.7). PRL-like B and I declined as lactation progressed (T mature: B, 41.74 +/- 8.9; I, 27.19 +/- 5.5; T late: B, 17.84 +/- 5.5; I, 27.33 +/- 1.8; PT mature: B, 59.85 +/- 16; I, 45.16 +/- 4.3). Milk PRL B to I ratios were consistently greater than serum B to I ratios during early lactation (milk: T, 1.4 +/- 0.3; PT, 3.6 +/- 1.3; serum: T, 1.0 +/- 0.2; PT, 0.58 +/- 0.12). During early lactation, high PRL-like B was widely distributed among several (n = 4-6) bioactive forms differing in molecular mass [8 to > 66 kilodaltons (kDa)] in T milk, but the majority of B in PT milk was detected in two or three forms. During mature and late lactation, lower PRL-like B was associated with two or three peaks (20 to > 66 kDa). A large fraction of PRL-like B (67%-84%) was associated with the phosphorylated (P-) fraction of human milk. Four immunoreactive forms (24, 30, 32, and 40 kDa) of P-PRL were identified by immunoblot analyses. Alkaline phosphatase treatment converted the 40-kDa immunoreactive P-PRL to 24-kDa PRL, increased the B of the P-fraction by 2-fold, but did not change total PRL I detected. PRL in the Concanavalin-A-retained fraction accounted for 59-69% of PRL in milk based on RIA results. No PRL-like B was detected in the Concanavalin-A-retained fraction of human milk; however, treatment of the glycosylated fraction of milk with peptide-N-glycosidase F increased thymidine incorporation by Nb2 cells 1.67-fold compared to that in controls. The results of this study show that human milk contains considerably greater PRL-like activity than previous reports based on RIA detection. The appearance and regulation of multiple bioactive PRL variants in milk throughout the course of lactation may serve as a mechanism by which milk PRL influences neonatal development.

Female↗

PubMed1995

Nature and bioactivity of gonadotropin-releasing hormone (GnRH) secreted during the GnRH surge.

Previous studies have demonstrated a neural action of estradiol in inducing a surge of GnRH in the ewe. However, although the GnRH and LH surges began concurrently, the GnRH surge consistently continued well beyond the surge of LH. Three experiments were conducted to test the hypothesis that the termination of the LH surge results from the secretion of a relatively inactive variant of GnRH during the later phases of the GnRH surge. In the first experiment, hypophyseal portal blood collected during an estrogen-induced LH surge was analyzed for GnRH immunoreactivity using two antibodies having specificity for the N- or C-terminal portion of the GnRH molecule. The duration, amplitude, and time course of the GnRH surge were found to be similar irrespective of the antisera used. In a second experiment, a competitive GnRH antagonist was administered at the beginning of the estrogen-induced GnRH/LH surge at a dose capable of blocking pituitary responsiveness for approximately half the duration of the GnRH surge. Antagonist treatment did not result in any change in the time of onset of the GnRH surge, but there was no increase in LH that naturally occurs coincident with onset of the GnRH surge. Rather, a persistent increase in LH secretion was observed during the latter stages of the GnRH surge, indicating that the GnRH molecules secreted at this time were biologically active. Finally, a sensitive and specific ovine pituitary cell bioassay was used to test bioactivity of GnRH in hypophyseal portal blood during different phases of the GnRH surge. GnRH bioactivity in samples collected early in the GnRH surge was greater than that before the onset of the GnRH surge but no greater than that collected during the descending limb of the surge. The results of all three experiments fail to support the hypothesis that the LH surge ends because of a change in the nature of the GnRH secreted. Rather they show that GnRH secreted throughout the surge is biologically active. Thus, the termination of the LH surge before that of the GnRH surge occurs for reasons other than lack of a bioactive GnRH signal.

Animals↗

PubMed1995

Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance.

Recombinant TSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of TSH by fusing the carboxy-terminal extension peptide (CTEP) of hCG beta onto TSH beta. When coexpressed either with alpha-subunit complementary DNA or alpha minigene in African green monkey (COS-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 25 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild-type (WT) and chimeric TSH secreted by COS-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the presence of terminal sialic acid and SO4 on their oligosaccharide chains, respectively. Chimeric and WT TSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [3H]thymidine incorporation. Chimeric TSH appears to be more effective in COS-7 cells than in 293 cells, as judged by growth assay. COS-7-produced chimeric TSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly beta and to a lesser extent alpha, appears to be responsible at least in part for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby, the in vivo bioactivity.

Animals↗

PubMed1996

The role of the asparagine-linked oligosaccharides of the alpha-subunit in human thyrotropin bioactivity.

TSH and the gonadotropins (FSH, LH, and hCG) are a family of heterodimeric proteins that share a common alpha-subunit and differ in their hormone-specific beta-subunit. The asparagine-linked (N-linked) oligosaccharides on these hormones are important in signal transduction. The N-linked oligosaccharides on the alpha-subunit have no effect on hCG and hFSH receptor binding, but are critical for their biological activity. Here, we analyzed the role of alpha-subunit N-linked oligosaccharides in human TSH (hTSH) bioactivity by site-directed mutagenesis and gene transfer. This was achieved by mutating the asparagine (Asn) residue in the N-linked glycosylation consensus sequence (Asn-X-Thr/Ser) to aspartic acid. The wild-type hTSH and its variants were expressed in Chinese hamster ovary cells. Wild-type alpha-subunit and its mutants (alpha 1, alpha 2, and alpha(1 + 2)) were efficiently combined with TSH beta-subunit and secreted as dimers. The bioactivity of TSH glycosylation variants was determined by measuring their abilities to stimulate cAMP formation and T3 secretion using a serum-free culture system of human thyroid follicles. Using this system, wild-type hTSH was significantly effective in the stimulation of cAMP formation and T3 secretion. Deletion of the oligosaccharide units from either site 1(alpha 1) or site 2(alpha 2) of the alpha-subunit increased the biological activity of the dimer by about 30%. However, deletion of carbohydrate units from both sites of hTSH alpha-subunit (alpha(1 + 2) resulted in a significant reduction in cAMP formation (by approximately 70%) and T3 secretion (by approximately 40%) compared to that with wild-type hTSH. These findings emphasize the importance of the alpha-subunit N-linked oligosaccharide chains on hTSH bioactivity.

Animals↗

PubMed1997

Recombinant human growth hormone-binding protein fails to enhance the in vivo bioactivity of human growth hormone in normal rats.

GH circulates in the plasma partially bound with a GH-binding protein (GHBP), but the physiological significance of the GHBP and how it affects GH bioactivity in vivo is still unknown. In the present study, we took advantage of the known biological action of exogenous human (h) GH to inhibit endogenous rat (r) pulsatile GH release and examined the effect of combining hGH with recombinant hGHBP on this response in normal rats. Spontaneous 7-h plasma rGH and hGH profiles were obtained from four groups of free-moving adult male rats s.c. administered either: 1) 200 microg hGH alone; 2) a mixture of 200 microg hGH and 200 microg hGHBP preincubated for 30 min before injection; 3) 200 microg hGHBP alone; or 4) Tris buffer (vehicle) alone. Rats administered the vehicle or hGHBP separately exhibited the typical pulsatile pattern of rGH secretion. Injection of hGH alone resulted in a marked (P < 0.01) suppression of spontaneous rGH pulses for approximately 3.5 h after the injection compared with vehicle-injected controls; during the subsequent 3.5- to 7-h period, recovery of spontaneous rGH peaks was evident. Plasma levels of hGH in these animals reached a peak within 1 h after hGH injection and declined to near undetectable levels by the end of the sampling period. In contrast, the disappearance rate of hGH was markedly slower in rats administered the hGH + hGHBP complex; plasma hGH concentrations at 7 h after injection were 14-fold higher than those in animals administered hGH alone, and hGH was still readily detectable up to 24 h after injection. However, despite the markedly higher levels of hGH persisting throughout the sampling period in complex-injected rats, both the time course of hGH-induced inhibition of rGH and the recovery of spontaneous rGH pulses were similar to those of animals administered hGH alone. Moreover, there were no significant modifications of plasma insulin-like growth factor-1 levels for up to 24 h after injection of the hGH + hGHBP complex. Computer simulations revealed that most of the total hGH observed during the 3.5- to 7-h period was circulating in the bound form. These results demonstrate that, despite hGHBP's ability to markedly prolong the bioavailability of hGH, precomplexing hGH with hGHBP failed to enhance hGH's in vivo bioactivity in the inhibition of endogenous pulsatile rGH release. Our findings do not provide support for the concept that the GHBP enhances the bioactivity of GH in vivo, at least over the time course examined here.

Animals↗

PubMed1976

Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and immunoreactive serum LH levels in normal subjects.

The effects of LHRH stimulation upon plasma LH levels measured by bioassay and radioimmunoassay were examined during the normal menstrual cycle, and in normal men and post-menopausal women. After administration of 100 mug LHRH by subcutaneous injection during the early follicular phase, a 2.7-fold rise in bioactive serum LH to 58 +/- 18 mIU/ml at 30-60 min was accompanied by an equivalent rise in immunoreactive LH, with unchanged bio:immuno (B:I) ratio of 0.9 +/ %/- 0.2 (SD). During the late follicular phase, bioactive serum LH rose 8-fold to 258 +/- 120 mIU/ml at 30-180 min, and the B:I ratio was significantly increased from 1.7 to 2.5. During the luteal phase, bioactive LH values rose 8.1-fold to 223 +/- 97 mIU/ml at 30-60 min, with increase in B:I ratio from 1.1 to 1.8. The LHRH-stimulated serum LH levels declined more rapidly in the early follicular phase than during the late follicular and luteal phases. Elevations of circulating LH concentrations following LHRH administration during the normal menstrual cycle were usually accompanied by a significant rise in B:I ratio, except during the early follic ular phase when the LH responses were small and the B:I ratio did not change. After LHRH stimulation of serum LH levels in men and post-menopausal women, relatively small and inconstant elevations of B:I ratio were observed above the basal value of 2.9. Although the B:I ratio was usually close to unity in cycling women, the ratio was from 2 to 3 in men and postmenopausal women, and during LHRH stimulation of normal women at the late follicular and luteal phases of the menstrual cycle. Thus, the increased LH secretion rate of post-menopausal women and LHRH-stimulated cycling women was frequently accompanied by a rise in the B:I ratio. These observations suggest that circulating LH molecules exhibit a relatively higher biological activity during states of increased biosynthesis and release of gonadotropins.

Adult↗

PubMed1985

The changing ratio of bioactive to immunoreactive luteinizing hormone (LH) through puberty principally reflects changing LH radioimmunoassay dose-response characteristics.

The ratio (B/I) of bioactive to immunoreactive LH in plasma varies during pubertal maturation. To elucidate the basis for these changes, we compared the dose-response characteristics of LH standards to those of plasma LH before and after GnRH infusion in normal males at various pubertal stages and girls with Turner's syndrome. We used a human LH (hLH) RIA modified to optimize specificity for LH bioactivity by employing a hLH tracer maximally bioactive in the rat interstitial cell testosterone production (RICT) bioassay. The pituitary hLH standards LER-907, NHPP I-1, and NHPP I-2 were not parallel to one another in the RIA, but were parallel in the RICT. Their relative slopes in the RIA were 1:1.35:1.49, respectively. The B/I for immunoreactive LH in these standards were 1 (LER-907):3 (I-1):5 (I-2). Dose-response characteristics varied greatly by patient category in the RIA. In contrast to the RICT, in which plasma from all subject groups, except the prepubertal basal group, gave parallel dose-response slopes, the groups differed in the steepness of their plasma LH RIA dose-response curves in the following order: adult post-GnRH congruent to adult basal congruent to pubertal post-GnRH greater than pubertal basal congruent to prepubertal post-GnRH congruent to prepubertal basal greater than Turner's syndrome. Prepubertal basal samples most closely resembled LER-907 in dose-response characteristics, while adult samples were most similar to NHPP I-1 and I-2. These characteristics were not related to the absolute LH concentration. Although the RIA dose-response characteristics of plasma LH changed during puberty, the B/I of basal LH did not increase through puberty using the modified hLH RIA, although B/I still rose in GnRH-stimulated samples during puberty. Our data demonstrate that the principal cause of variability in B/I is the changing dose-response characteristics of plasma LH in the RIA. We suggest that the source of this variability is a change in the molecular characteristics of LH in different states of pubertal maturation and gonadal function. The results imply that better ways of assaying LH are needed.

Adolescent↗

PubMed1988

Bioactive follicle-stimulating hormone responses to intravenous gonadotropin-releasing hormone in boys with idiopathic hypogonadotropic hypogonadism.

To test the hypothesis that exogenous pulsatile administration of GnRH will increase serum bioactive FSH (bFSH) levels, we studied four boys with suspected idiopathic hypogonadotropic hypogonadism (IHH). These boys presumably secreted relatively little GnRH. By virtue of their low baseline serum gonadotropin levels yet responsive pituitary gonadotrophs, these boys with IHH proved to be an excellent clinical model to test this hypothesis. Administration of GnRH (0.025 microgram/kg.dose) iv at 1- or 2-h intervals for 3-5 days resulted in an increase in serum bFSH after 91% of the GnRH doses. Serum immunoreactive FSH (iFSH) and LH (iLH) levels increased after 42% and 64% of the GnRH doses, respectively. Ninety percent of the iLH responses were concordant with bFSH responses, but only 33% of the iLH responses were concordant with iFSH responses. The serum bFSH responses occurred consistently within 20 min after GnRH administration and resulted in an increased serum bioactive to immunoreactive FSH ratio. By 60 min, serum bFSH levels had returned to preinjection levels. Serum testosterone and estradiol levels did not change during the period of GnRH administration in three of the four boys. We conclude that pulsatile, low dose iv GnRH administration in boys with IHH elicits significant serum bFSH increases by 20 min; the newly secreted FSH is preferentially enriched with increased in vitro FSH bioactivity, and it is rapidly cleared from serum (60 min). Therefore, serum bFSH measurements may provide a sensitive index of GnRH effects on the gonadotrophs.

Adolescent↗