Pegylated peptides. IV. Enhanced biological activity of site-directed pegylated GRF analogs

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.     

New Gaba-containing analogues of human growth hormone-releasing hormone (1-30)-amide: I. Synthesis and in vitro biological activity

BACKGROUND AND METHODS: Congenital cystic dilatation of the bile ducts represents an uncommon anomaly of the biliary system. We report on 6 patients suffering from cystic biliary duct dilatations which were treated in our hospital between 1980 and 1992. Clinical signs included upper abdominal pain, white or clay-colored stool, icterus and/or palpable tumor. According to the classification of Todani, 4 children had type Ia cysts, 1 child a type Va cyst and 1 child a type Ia cyst with extrahepatic biliary atresia. RESULTS: Among the diagnostic methods sonography is preeminent and permitted demonstration of intra- and extrahepatic biliary duct dilatations in all of our patients. In 2 patients small cystic dilatations could be distinguished from hepatic vessels by colour-coded Doppler sonography. CONCLUSION: The treatment of choice is the resection of the dilated extrahepatic biliary ducts followed by hepaticojejunostomy using the Roux-en-Y-technique.     

Feeding effects of growth hormone-releasing factor in rats are photoperiod sensitive.

By examining the influence of photoperiod, this study extended previous findings showing that centrally administered growth hormone-releasing factor (GRF) increases feeding. Rats received icv injections of GRF (0, 0.4, 4.0, and 40.0 pmol) during either the light or dark phase of the photocycle. GRF produced a dose-dependent increase in 90-min food intake during the light photoperiod but a dose-dependent suppression during the dark photoperiod. GRF did not significantly alter 24-hr feeding or locomotor activity, and a biologically inactive analog of GRF had no effect on feeding. Possible mechanisms underlying GRF's photoperiod dependency are discussed, including a suggestion that endogenous GRF may play a role in the regulation of circadian feeding. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Early biological effect of in vivo gene transfer of platelet-derived growth factor (PDGF)-B into healing patellar ligament

To define the early biological effect of in vivo introduction of the PDGF-B gene on the healing of ligaments, a HVJ-liposome suspension containing platelet-derived growth factor (PDGF)-B cDNA was injected directly into the injured patellar ligament of 14-week-old male Wistar rats. Rats were killed at 1, 4 and 8 weeks for the morphological analysis of angiogenesis by laminin immunohistochemistry and of collagen deposition by Masson's Trichrome staining and collagen I immunohistochemistry. PDGF-B gene transfer caused the enhanced expression of PDGF in healing ligament up to 4 weeks after transfection, leading to an initial promotion of angiogenesis and subsequent enhanced collagen deposition in the wound. Enhanced and accelerated matrix synthesis in the PDGF-B gene introduced healing ligament suggests that this gene transfer technique may be a potentially useful tool for improving soft tissue repair.     

Circular dichroism (CD) studies on biological activity of mast cell degranulating (MCD) peptide analogs

Analogs of MCD peptide were synthesized by solid-phase methods. Positive charges were deleted at the N-and/or C-terminus, including the helical portion of the molecule. Four peptides were prepared by removing residues 16-18 (Arg-Lys-Ile), 1-2 (Lys), 1-2 and 16-18 and by acetylation of the amino end (Ile). Analogs were tested on mast cells for histamine-releasing activity. Although the helicity of these derivatives, determined by circular dichroism (CD), was not significantly different from the native MCD peptide, two analogs with C-terminal deletions showed a 5- to 10-fold decrease in activity. These findings suggest that the C-terminus is more important than the N-terminus in determining bioactivity of MCD peptide.     

Proteolytic degradation of rat growth hormone-releasing factor(1-29) amide in rat pituitary and hypothalamus

The identification of peptide bonds vulnerable to tissue peptidases is a valuable approach to design peptide agonists which exhibit a longer duration of action than the native molecules. Therefore, the kinetic of disappearance of rat growth hormone-releasing factor (rGRF(1-29)NH2) and the identification of its metabolites were studied in rat pituitary and hypothalamus. Synthetic rGRF(1-29)NH2 (10 microM) was incubated (0-120 min, 37 degrees C) in the presence of a pituitary (237 +/- 51 micrograms protein/ml) or hypothalamus homogenate (576 +/- 27 micrograms protein/ml). Using analytical high pressure liquid chromatography (HPLC), apparent half-lives of 22 +/- 3 min and 25 +/- 4 min were found in pituitary and hypothalamus, respectively. In both tissues, three degradation products, all less hydrophobic than the native peptide, were detected and isolated by preparative HPLC. The identification of the purified metabolites was ascertained by amino acid analysis, sequencing and chromatography with synthetic homologs. These results indicate that the main sites of cleavage in the pituitary and hypothalamus are Lys21-Leu22 (trypsin-like cleavage site), Leu14-Gly15 and Tyr10-Arg11 (chymotrypsin-like cleavage sites). TLCK and leupeptin did not affect the formation of fragment (1-21)OH while TPCK blocked the cleavage of Leu14-Gly15. The low affinity of fragment (1-21)NH2 for pituitary GRF binding sites suggests that hydrolysis of the Lys21-Leu22 bond inactivates rGRF(1-29)NH2 in this target tissue.     

Direct effects of growth hormone (GH)-releasing hexapeptide (GHRP-6) and GH-releasing factor (GRF) on GH secretion from cultured porcine somatotropes

Growth hormone (GH)-releasing hexapeptide (GHRP-6) belongs to the expanding family of synthetic GH secretagogues (GHSs). Previous studies have shown that non-peptidyl GHRP-6 analogues stimulate GH release in vivo in pigs, and interact synergistically with GH-releasing factor (GRF), but its direct effects on porcine somatotropes have not been addressed hitherto. In the present study, we have evaluated the response of cultured porcine pituitary cells to GHRP-6, and its interaction with GRF and somatostatin (SRIF). Secretory response of somatotropes was assessed by using two distinct techniques. GH released by monolayer cell cultures was evaluated by enzyme immunoassay, whereas that secreted by individual somatotropes was measured by immunodensitometry using a cell blotting assay. Our results demonstrate that both GHRP-6 and GRF stimulated GH release from monolayer cultures at doses equal to or above 10(-9) M. Use of cell immunoblot assay demonstrated that, like GRF, the hexapeptide acts directly upon porcine somatotropes to exert its action. Moreover, regardless of the technique applied, combined administration of GHRP-6 (10(-6) or 10(-9) M) and GRF (10(-8) M) resulted in an additive, but not synergistic, stimulatory GH response. Finally, SRIF (10(-7) M) inhibited the stimulatory effect of GHRP-6 alone or in combination with GRF. These results indicate that GHRP-6 directly and effectively stimulates GH secretion from porcine somatotropes in vitro, and acts additively when coadministered with GRF. Therefore, the synergistic stimulatory effect of GHSs and GRF reported in vivo in this species might require additional factors that are lacking in the in vitro situation.     

Embryonic origin of preimplantation factor (PIF): biological activity and partial characterization

Preimplantation factor (PIF) is detected in the serum of women shortly after fertilization; its origin, however, has not been established. In this study, the embryonal origin of PIF was investigated and partial characterization of the factor was carried out. Culture media from viable human 2-8-cell stage embryos and mouse 2-cell-blastocyst stage embryos were analysed using the lymphocyte/platelet binding assay (LPBA). The assay was performed by combining culture media with donor O+ type blood-derived lymphocytes/platelets, complement and an antibody against CD2. Increased autorosette formation between lymphocytes and platelets (> 9%) was an indication for the presence of PIF. In addition, the effect of platelet-activating factor (PAF) and chaperonin 10 on PIF activity was determined. Partial purification of PIF was carried out using gel filtration and reverse-phase high purification liquid chromatography (HPLC), followed by mass spectrometry. Culture media of single human viable fertilized oocytes were negative for PIF; however, the 10-fold concentrated medium was positive for PIF. In medium in which five or more mouse embryos were cultured, PIF activity was observed starting at the morula stage and was higher by the blastocyst stage. Addition of PAF or chaperonin 10 to the PIF assay did not elicit a specific effect on PIF activity. Chromatographic data suggest that PIF activity is due to low molecular weight proteins. PIF appears to be a low molecular weight protein which is derived from viable preimplantation embryos. It is different from PAF or chaperonin 10. Its final characterization will be valuable for better understanding of maternal recognition of pregnancy and implantation.     

alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38-61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-alphavbeta3 antibodies prevent cell adhesion to FGF-2. Also, purified human alphavbeta3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-alphavbeta3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with alphavbeta3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.     

Luteinizing hormone-releasing hormone agonists interfere with the mitogenic activity of the insulin-like growth factor system in androgen-independent prostate cancer cells

We have previously shown that LHRH agonists exert a direct and specific inhibitory action on the proliferation of the androgen-independent DU 145 prostate cancer cell line; however, the cellular mechanisms mediating this antiproliferative action are not well defined. It is well known that the insulin-like growth factor (IGF) system plays a crucial role in the local regulation of the growth of androgen-independent prostate cancer. The present experiments were performed to evaluate whether LHRH agonists might exert their antimitogenic effect by interfering with the activity of the locally expressed IGF system. To this purpose, the effects of the LHRH agonist Zoladex (LHRH-A) on 1) the mitogenic action of IGF-I, 2) the tyrosine phosphorylation of type 1 IGF-I receptor (IGF-IR), 3) the concentration of IGF-IR, and 4) the secretion of IGF-binding protein-3 were studied. The results obtained show that in DU 145 cells, LHRH-A 1) counteracts the mitogenic action of IGF-I in a dose-dependent manner, 2) prevents the IGF-I-induced tyrosine phosphorylation of the IGF-IR, 3) reduces the concentration of IGF-IR without affecting its Kd value, and 4) does not affect the secretion of IGF-binding protein-3 in the conditioned medium from these cells. These data suggest that LHRH agonists may inhibit the proliferation of human androgen-independent prostate tumor cells by interfering with some of the cellular mechanisms mediating the stimulatory action of the IGF system.     

Transforming growth factor beta (TGF beta) activity in urine of patients with glomerulonephritis

Several lines of experimental evidence have shown that transforming growth factor beta (TGF beta) may play major role in glomerular diseases, mediating the inflammatory response through glomerulosclerosis. In the present study we evaluated TGF beta activity in occasional urine samples from 7 normal individuals and from 15 patients (10 with focal glomerular sclerosis and 5 with membranous glomerulonephritis) using a CCL-64 mink lung cell growth inhibition assay. Urinary TGF beta activity (reported in relation to urine creatinine concentration, Ucr, mean +/- SD) was higher in patients with focal glomerular sclerosis (mean = 17.32 +/- 15.75/10 micrograms Ucr) and patients with membranous glomerulonephritis (mean = 17.78 +/- 11.53/10 micrograms Ucr) than in normal individuals (mean = 0.8 +/- 0.44/10 micrograms Ucr). We also observed that TGF beta activity in urine from patients with focal glomerular sclerosis correlated with their plasma creatinine levels (r = 0.85), suggesting that TGF beta activity may be correlated with other indices of disease progression. Our data suggest that measurement of urinary TGF beta activity could be a useful noninvasive procedure for the evaluation of renal TGF beta production, which may be useful to assess prognosis and to evaluate therapeutic efficacy in patients with renal disease.     

Chemical synthesis and biological activities of the EGF-like domain of mouse schwannoma-derived growth factor (SDGF)

Schwannoma-derived growth factor (SDGF), an epidermal growth factor (EGF) family peptide recently discovered, has an EGF-like domain in the carboxyl terminal portion. In this study, we synthesized mSDGF(38-80) corresponding to the EGF-like domain of mouse SDGF by means of stepwise solid-phase method using Fmoc chemistry in order to evaluate the biological function of the EGF-like domain in SDGF. The linear peptide of mSDGF(38-80) was folded by direct oxidation with reduced and oxidized glutathione to form intramolecular disulfide bridges in synthetic peptide. On the biological activity, we examined mitogenic activity induced by mSDGF(38-80) in NIH/3T3 fibroblast cells and interaction with EGF receptor in A431 cells. In the results, mSDGF(38-80) was confirmed to form three disulfide linkages that were similar in pattern to EGF by amino acid and sequence analysis of fragments obtained after thermolytic digestion. However, mSDGF(38-80) possessed weak mitogenic activity in NIH/3T3 cells and weak binding affinity for the EGF receptor in A431 cells compared with those of human EGF. These results suggest that the EGF-like domain of SDGF may have little effect upon mitogenic activity and the EGF receptor binding of SDGF.     

Splenic primitive hematopoietic stem cell (PHSC) activity is enhanced by steel factor because of PHSC proliferation

To test whether primitive hematopoietic stem cells (PHSCs) are stimulated by Steel (SI) factor (c-kit ligand) in vivo, donor mice were studied after three or seven daily injections of SI factor. PHSC activity was measured as long-term erythroid and lymphoid competitive repopulating ability. Cells to be tested (usually marrow or spleen cells from treated donors) were mixed with untreated competitor marrow that produces erythrocytes and lymphocytes that are genetically distinguishable from the donors by differences in hemoglobin (Hb) and glucosephosphate isomerase (GPI) markers. These cell mixtures were injected into lethally irradiated hosts, and after 111 to 293 days, functional abilities of donor PHSC populations were assessed and expressed as percentages of donor-type Hb and GPI in the host's circulating erythrocytes and lymphocytes, respectively. A striking increase in splenic PHSC activity occurred after seven daily injections of SI factor, with a much smaller increase after three daily injections. Both three and seven daily injections of SI factor slightly reduced marrow PHSC activity. Rapid cycling greatly increases PHSC vulnerability to 5-fluorouracil (5FU). To test whether SI factor stimulates PHSCs into rapid cycling, donor mice were given a dose of 5FU in addition to SI factor. The increase in splenic PHSCs after 7 days of treatment with SI factor occurred to a similar degree whether donors were or were not treated with 5FU on day 8. However, a dose of 5FU on day 4 of the SI factor treatments almost totally prevented the increase in splenic PHSC activity. Apparently this increased activity requires PHSC cycling throughout the period of SI factor treatment.     

Effects of growth hormone-releasing factor and(or) thyrotropin-releasing hormone on growth, feed efficiency, carcass characteristics, and blood hormones and metabolites in beef heifers

The objective of this study was to determine the effect of long-term administration of a growth hormone (GH)-releasing factor analog (GRFa) and(or) thyrotropin-releasing hormone (TRH) on growth, feed efficiency, carcass characteristics, and blood hormones and metabolites in beef heifers. Crossbred heifers (n = 48; 345.9 +/- 2.8 kg) were divided into four equal groups: control (vehicle), 1 microgram of GRFa (human GRF 1-29 analog).kg BW-1.d-1, 1 microgram of TRH.kg BW-1.d-1, or GRFa + TRH. Daily s.c. injections continued for 86 d. Blood samples were collected from half of the heifers after injection on d 1, 36, and 78. On d 89, all heifers were slaughtered. Treatments did not affect (P > .05) ADG but GRFa + TRH decreased (P < .05) ADFI relative to all other treatments. Feed conversion efficiency tended (P < .10) to be improved in the groups given GRFa alone or TRH alone. Treatment with GRFa and(or) TRH did not affect carcass weight, dressing percentage, conformation score, backfat thickness, or weights of liver, kidneys, pituitary, and ovaries. The GRFa + TRH treatment reduced (P < .05) fat score and increased (P < .05) longissimus muscle area relative to other treatments. The GRFa treatments reduced (P < .05) the weight and fat percentage of the mammary gland and increased (P < .05) heart weight. Treatment with TRH alone failed to stimulate GH on d 1, 36, and 78. Treatment with GRFa alone increased (P < .05) GH above controls on d 36, whereas GRFa + TRH increased (P < .05) GH on d 1, 36, and 78. Treatment with GRFa alone increased (P < .05) IGF-I only on d 1, whereas GRFa + TRH was without effect on all days. Across sampling days, treatments had little effect on blood concentrations of insulin, triiodothyronine, nonesterified fatty acids, urea nitrogen, and glucose. The GRFa alone and GRFa + TRH decreased (P < .05) and TRH alone increased (P < .05) thyroxine concentrations. In conclusion, with the dose and administration regimen used, GRFa and(or) TRH yielded small but positive improvements in animal performance.     

Design and evaluation of radioactive acetylcholine analogs for mapping brain acetylcholinesterase (AchE) in vivo

For mapping brain acetylcholinesterase (AchE) in vivo, seven radioactive acetylcholine analogs, N-[14C]methylpiperidyl-3- and 4-acetates, propionates, isobutyrates, and 3-butyrate were newly synthesized and evaluated in mice. The esters readily entered the brain and were hydrolyzed into the hydrophilic metabolite, which was trapped. In brain homogenates, the esters showed a wide range of enzymatic reactivity (about 40-fold), and high specificity for AchE (more than 82%) except the butyrate. Intra-brain distribution of the esters reflected a pattern of AchE activity.     

Effects of arginine, growth hormone-releasing hormone (GHRH) and neostigmine administered singly or in paired combinations on growth hormone (GH) release in pigs

OBJECTIVE: A multicentre study was undertaken to determine the value of somatostatin receptor (sst) scintigraphy in predicting hormonal and visual responses to octreotide treatment in GH-secreting and non-functioning pituitary adenomas. SUBJECTS AND METHODS: Somatostatin receptor scintigraphy was performed in 48 patients (19 acromegaly, 29 non-functioning pituitary adenomas with ophthalmological defects). Results were expressed as an uptake index of the pituitary area. A threshold for positivity was determined in 23 subjects considered as controls. Thirty-five patients were treated for 1 month with octreotide (300 micrograms daily). The therapeutic response was assessed on GH and IGF-I suppression or evolution of the ophthalmological defects. The relationships between the somatostatin receptor scintigraphy result, the therapeutic effect of octreotide and in vitro studies performed in 12 tumours were studied. RESULTS: From the results of control subjects the uptake index threshold for positivity was 2. In patients, somatostatin receptor scintigraphy was positive in 64% and there was no relationship between uptake index and tumour size. In GH tumours, somatostatin receptor scintigraphy was positive in 68%; uptake index was related to octreotide-induced GH and IGF I suppression. The positive predictive value was 100% and the negative predictive value was 50%. In vitro studies showed detectable binding sites for somatostatin with sst2 and sst5 expression in the 4 GH tumours studied although somatostatin receptor scintigraphy was negative in 2 cases. In non-functioning pituitary adenomas somatostatin receptor scintigraphy was positive in 62%. Based on visual effects, the positive predictive value was 61% and the negative predictive value was 100%. A wide distribution of somatostatin binding sites was found in 8 non-functioning pituitary adenomas with expression of sst2 only. CONCLUSION: In the conditions of the study, in patients with acromegaly, positive somatostatin receptor scintigraphy predicts a hormonal response but the value of somatostatin receptor scintigraphy is limited by its low negative predictive value. In patients with non-functioning pituitary adenomas, negative somatostatin receptor scintigraphy predicts that there will be no visual improvement during octreotide treatment.     

Astressin analogues (corticotropin-releasing factor antagonists) with extended duration of action in the rat

In earlier reports we identified specific point substitutions (DPhe12,Nle21,38), cyclization strategies [in particular, introduction of lactam rings such as that of cyclo(Glu30,Lys33)], and deletions (residues 1-7) in the CRF molecule that led to agonists. We also noted that further deletions (residues 8-14) produced antagonists such as astressin ?cyclo(30-33)[DPhe12,Nle21,38, Glu30, Lys33]hCRF(12-41)? (1). We hypothesized that the lactam ring promoted conformational stability to yield analogues with increased potency both in vitro and in vivo as compared to that of their linear counterparts. Additionally, we reported that cyclo(30-33)[DPhe12,Nle21,38, Glu30,DHis32,Lys33]hCRF(12-41) (3) and dicyclo(26-36,30-33)[Ac-Asp9,DPhe12,Nle21,38, Cys26, Glu30,Lys33, Cys36]hCRF(9-41) were ca. twice and 1/100 as potent as astressin, respectively, suggesting a putative turn that encompasses residues 30-33 (previous paper: Koerber et al. J. Med. Chem. 1998, 41). To increase the potency of 1 and/or 3 in vivo, we extended their chain length by one (5-8), two (9, 10), and three (11, 12) residues at the N-terminus and acetylated (6, 8, 10, 12). Of the compounds tested for duration of action (1, 3-6, 8), we found 6 and 8 to be slightly longer-acting than astressin or [DHis32]astressin, while their potencies in vitro were not significantly different from that of 3. Additionally, we introduced CalphaMe-leucine residues in lieu of leucine at positions 14, 15, 19, 27, and 37 in [DHis32]astressin. The analogue [CalphaMe-Leu27,DHis32]astressin (16) was more potent (although not statistically in all cases) than the other four analogues in vitro. While acetylation of the N-terminus of 16 (i.e., 18) or of [CalphaMe-Leu27]astressin (i.e., 19) did not have a significant effect on in vitro potency, elongation of the N-terminus by one or three residues in addition to acetylation resulted in cyclo(30-33)[DPhe12,Nle21,CalphaMe-Leu27,Glu3 0,DHis32,Lys33, Nle38]Ac-hCRF(11-41) (21), cyclo(30-33)[DPhe12,Nle21,CalphaMe-Leu27, Glu30,Lys33,Nle38]Ac-hCRF(9-41) (22), and cyclo(30-33)[DPhe12, Nle21, CalphaMe-Leu27,Glu30,DHis32,Lys33,Nle38 ]Ac-hCRF(9-41) (23) that were longer-acting than 6 and 8 (ca. 2 h inhibition of ACTH secretion at 25 micrograms/adrenalectomized rat). Analogues 22 and 23 were also more potent than astressin at reversing intracisternal CRF- and abdominal surgery-induced delay of gastric emptying in conscious rats.     

The prevalence of factor V Leiden (1691 G-->A) mutation in Turkey

Resistance to activated protein C (APC), which is caused by a single point mutation in the gene for factor V, is a common risk factor for thrombosis. In this study, we screened factor V (FV) Leiden mutation in 81 subjects. The mutation in the heterozygous form was found in 7.1 percent of our normal population. This high frequency suggests that screening for the FV mutation should be considered in patients with a family history of thrombosis.     

Insulin-like growth factor I is an independent coregulatory modulator of natural killer (NK) cell activity

We aimed to investigate the natural killer (NK) cell activity in hGH-deficient adults and to analyze the effect of insulin-like growth factor (IGF)-I in vivo and in vitro on NK cell activity. NK cell activity was measured in a 4-h nonisotopic assay with europium-labeled and cryopreserved K-562 cells. NK-cell numbers were measured after incubation with murine monoclonal CD3 and CD16 antibodies by flow cytometry analysis. In a cross-sectional study, the basal and interferon-beta (IFN-beta) stimulated (1000 IU/ml) NK cell activity of 15 hGH-deficient patients and 15 age- and sex-matched controls was measured. The percentages and absolute numbers of CD3-/16+ NK-cells were not significantly different in the patient vs. control group. The basal and IFN-beta stimulated NK cell activity however was significantly decreased in the patient vs. control group at all effector/target (E/T) cell ratios from 12.5-100 (e.g. 17 +/- 3 vs. 28 +/- 3% lysis without IFN-beta, P < 0.05, and 42 +/- 4 vs. 57 +/- 4% lysis with IFN-beta, P < 0.05; both at E/T 50). IGF-I levels of patients and controls showed a significant positive correlation with NK cell activity (r = 0.37; P < 0.05). In an IGF-I in vitro study (IGF-I in vitro 250-1250 microg/L), the basal and IFN-beta stimulated NK cell activity of 13 hGH-deficient patients and of 18 normal subjects was significantly enhanced by IGF-I in vitro (e.g. GH-deficient patients: 9 +/- 2 vs. 10 +/- 2% lysis without IFN-beta, P < 0.05 and 25 +/- 4 vs. 30 +/- 4% lysis with IFN-beta, P < 0.005; and normal subjects: 15 +/- 3 vs. 23 +/- 3% lysis without IFN-beta, P < 0.001 and 35 +/- 4 vs. 44 +/- 5% lysis with IFN-beta, P < 0.001; both at IGF-I 500 microg/L). In summary, in our cross-sectional study, adult GH-deficient patients showed a significantly lower basal and IFN-beta stimulated NK cell activity than matched controls, despite equal NK cell numbers. IGF-I levels of patients and controls showed a weak positive correlation with NK cell activity. In an in vitro study, IGF-I significantly enhanced basal and IFN-beta stimulated NK cell activity of hGH-deficient patients and also of normal subjects. The decreased NK cell activity in GH-deficient patients may be caused at least in part by low serum IGF-I levels. IGF-I appears to be an independent coregulatory modulator of NK cell activity.     

Longtime administration of growth hormone-releasing hormone (GHRH) does not restore the reduced efficiency of GHRH on sleep endocrine activity in 2 old-aged subjects--a preliminary study

Aging results in a more shallow sleep accompanied by a blunted growth hormone (GH) secretion. In young male normal controls repetitive administration of GH-releasing hormone (GHRH) at the beginning of the night results in an increased secretion of GH, a blunting of cortisol and a stimulation of slow-wave sleep (SWS). In healthy elderly men and women, however, GHRH exerts only weak effects on sleep-endocrine activity. In a previous report continuous treatment of healthy elderly males by repetitive administration of GHRH (during 12 days administration with 100 micrograms GHRH i.v. at 9.00 h every second day, "priming") enhanced GHRH stimulated GH secretion at daytime markedly. We tested if priming with GHRH results in a more distinct modulation of the nocturnal hormone secretion and of the sleep EEG than acute administration of the peptide. Two elderly male controls spent first three consecutive nights in the sleep laboratory, the first of which served for adaptation to laboratory conditions. During the two other nights (at days 1 and 2) sleep EEG was recorded and blood was sampled for determining the secretion of GH, cortisol and ACTH. In one of the nights the subjects received 50 micrograms GHRH hourly between 22.00 h and 1.00 h (4 x 50 micrograms) or placebo. The next examination followed after the priming period at day 14 and the last was performed two weeks after treatment at day 28. After the baseline administration of 4 x 50 micrograms GHRH before priming no clear changes of sleep EEG towards improved sleep were detectable, whereas GH secretion was increased. After priming sleep period time and SWS time were lower compared to the baseline night with GHRH administration, whereas REM time duration increased. GHRH induced GH secretion was not enhanced after priming. ACTH secretion was markedly enhanced compared to baseline stimulation. We conclude that priming with GHRH has no sleep improving effect and does not change hormone secretion in elderly normal subjects. Hence in the elderly priming with GHRH is not capable to induce a rejuvenation of sleep endocrine activity.