Lack of association between allelic status and myostatin content in lambs with the myostatin g+6723G>A allele.

Lambs with the myostatin (MSTN) g+6723G>A mutation have a greater muscle mass, which is believed to be associated with reduced myostatin protein abundance. This experiment was designed to determine if differences in allelic frequency of the MSTN g+6723G>A mutation affected abundance of myostatin protein from birth to 24 wk of age. A Poll Dorset cross White Suffolk ram (MSTN A/G) was mated to 35 White Suffolk cross Border Leicester cross Merino ewes (MSTN A/G, n=21, and MSTN G/G, n=14). The progeny of these matings delivered 44 lambs with MSTN A/A (n=9), MSTN A/G (n=21), and MSTN G/G (n=14) genotypes. At approximately 1, 4, and 12 wk of age, a biopsy sample was collected and a blood sample was taken to measure the abundance of myostatin protein in muscle and plasma. At approximately 24 wk of age, the wether lambs were slaughtered to determine carcass characteristics and muscle samples were taken from the bicep femoris. The abundance of mature myostatin protein in muscle from 1 wk old lambs was less (P=0.05) in MSTN A/A and MSTN A/G compared with MSTN G/G lambs. However, at 4 and 24 wk the MSTN A/A lambs had a greater (P=0.04) abundance of myostatin protein compared with the MSTN A/G and MSTN G/G lambs. The abundance of mature myostatin did not differ between genotypes in plasma but the myostatin protein did increase as the lambs aged. At slaughter the MSTN A/A wether lambs had greater dressing percentages (P=0.04), shortloin (P=0.01), topside (P<0.001), and round (P=0.01) weights but did not differ in final BW or HCW (P>0.05). The MSTN A/A lambs had more muscle fibers (P=0.02) in the cross-section of LM between the 12th and 13th rib. The MSTN A/A lambs also had greater lean (P=0.002), less fat (P=0.009), and reduced organ (heart, liver, spleen, and kidneys) mass as determined by computed tomography scanning than MSTN G/G lambs. The results of this study demonstrated that lambs homozygous for the MSTN g+6723G>A mutation have changes in carcass characteristics (dressing and total lean), organ weights, and muscle fiber number. This may be due to reduced myostatin protein early in utero, but after 4 wk of age there was no difference in the abundance of mature myostatin protein in muscle or plasma among MSTN A/A, MSTN A/G, and MSTN G/G genotypes.

Microbiology of spent nuclear fuel storage basins.

Microbiological studies of spent nuclear fuel storage basins at Savannah River Site (SRS) were performed as a preliminary step to elucidate the potential for microbial-influenced corrosion (MIC) in these facilities. Total direct counts and culturable counts performed during a 2-year period indicated microbial densities of 10(4) to 10(7) cells/ml in water samples and on submerged metal coupons collected from these basins. Bacterial communities present in the basin transformed between 15% and 89% of the compounds present in Biologtrade mark plates. Additionally, the presence of several biocorrosion-relevant microbial groups (i.e., sulfate-reducing bacteria and acid-producing bacteria) was detected with commercially available test kits. Scanning electron microscopy and X-ray spectra analysis of osmium tetroxide-stained coupons demonstrated the development of microbial biofilm communities on some metal coupons submerged for 3 weeks in storage basins. After 12 months, coupons were fully covered by biofilms, with some deterioration of the coupon surface evident at the microscopical level. These results suggest that, despite the oligotrophic and radiological environment of the SRS storage basins and the active water deionization treatments commonly applied to prevent electrochemical corrosion in these facilities, these conditions do not prevent microbial colonization and survival. Such microbial densities and wide diversity of carbon source utilization reflect the ability of the microbial populations to adapt to these environments. The presumptive presence of sulfate-reducing bacteria and acid-producing bacteria and the development of biofilms on submerged coupons indicated that an environment for MIC of metal components in the storage basins may occur. However, to date, there has been no indication or evidence of MIC in the basins. Basin chemistry control and corrosion surveillance programs instituted several years ago have substantially abated all corrosion mechanisms.

Laparoscopic resection of an ovarian pregnancy in a patient using the copper T intrauterine device.

This is a case report of a 40-year-old multiparous woman with a right ovarian pregnancy. The patient has had a copper T intrauterine device for about 6 years. The diagnosis of ovarian pregnancy was based on the improper rise of serum beta-hCG levels, sonographic findings of an empty uterus, and the laparoscopic verification of Spiegelberg's criteria. The treatment was by laparoscopic resection of part of the right ovary with the pregnancy. Pathological examination confirmed the presence of ovarian tissue around the chorionic villi. The relationship between the use of the intrauterine device and ovarian pregnancy is discussed.

Evidence for suppression of immune function by insulin-like growth factor-1 in dwarf rats in vivo.

These studies were undertaken to investigate the effects of increasing or decreasing IGF-1 levels on aspects of immune function in rats. Female dwarf rats were treated with recombinant human IGF-1 or with a potent sheep anti-IGF-serum. Body weight, thymus weight and spleen weight increased with IGF-1 treatment (p < 0.001), while there was no effect of anti-IGF-1 treatment when compared with the appropriate normal sheep serum (NSS) treated controls. IGF-1 treatment significantly decreased WBC and RBC counts, but increased the ratio of CD4+:CD8+ T-cells. Anti-IGF-1 serum had no effect on these parameters compared with NSS. However anti-IGF-1 was associated with increased T-cell numbers, decreased natural killer cells, and enhancement of the animals' ability to produce specific IgG in response to injection of keyhole limpet haemocyanin (KLH). These results indicate that IGF-1 may suppress immune function although increasing the size of immune organs such as spleen.

New alpha-thiol dipeptide dual inhibitors of angiotensin-I converting enzyme and neutral endopeptidase EC 3.4.24.11.

Dual inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP, EC 3.4.24.11) and angiotensin-I converting enzyme, have been the focus of much clinical interest for the treatment of hypertension and congestive heart failure. A novel series of alpha-thio dipeptides containing central cyclic non-natural amino acids were prepared and were evaluated for their ability to inhibit these two metallopeptidases in vitro and in vivo. Most of these compounds were found to be excellent dual inhibitors of ACE and NEP in vitro and several were also found to inhibit angiotensin-I (AI) pressor response in conscious rats when given by intravenous administration. Compound 6n, one of our most potent dual inhibitors in vitro, was found to be more efficacious than captopril in the AI pressor experiment when administered orally to conscious rats. This compound was also found to inhibit plasma NEP activity following oral administration to conscious rats and was more efficacious than acetorphan. The structure-activity relationships and biological activity of these dual inhibitors will be discussed.

Growth hormone alters lymphocyte sub-populations and antibody production in dwarf rats in vivo.

Female dwarf rats at different ages were treated with recombinant porcine GH or with a potent sheep anti-rat GH serum. Body weight and spleen weight increased with GH and decreased with anti-GH treatment (p < 0.001). Neither GH nor anti-GH treatment resulted in a change in circulating WBCs, but GH decreased, while anti-GH increased, RBC counts (p < 0.001). Similarly, GH treatment tended to decrease the ratio of CD4+:CD8+ T-cells while anti-GH increased (p < 0.05) the ratio. Anti-GH treatment also enhanced the animals' ability to produce specific IgG in response to KLH injection. These results indicate that GH may have a physiological role in suppressing humoral immune function but may enhance cell-mediated immunity.

Neuroendocrine regulation of growth hormone secretion in sheep. VII. Effects of GABA.

The effects of intravenous (i.v.) or intracerebroventricular (i.c.v.) administration of gamma aminobutyric acid (GABA) on plasma growth hormone (GH) concentrations have been examined in sheep. Intravenous administration of GABA resulted in a rapid, significant (P < 0.001) increase in plasma GH. Administration of 10 mg of GABA i.c.v. produced a significant (P < 0.001) increase in GH release. By contrast, 100 mg given i.c.v. was inhibitory and resulted in a decrease (P < 0.05) in plasma GH levels. Concurrent administration of somatostatin (0.5 microgram/min i.v. over 1 h) did not alter the plasma GH response to 10 mg GABA given i.c.v. These data are consistent with the concept of dual sites of action for GABA in regulating GH release in sheep, but the exact mechanism through which this effect is mediated remains unclear.

Alteration of maternal growth hormone levels during pregnancy influences both fetal and postnatal growth in rats.

To study the effects of exogenous growth hormone (GH) administration to the mother on fetal growth, dwarf rats with an isolated GH deficiency were given daily s.c. injections of GH throughout pregnancy. Fetuses were heavier in GH-treated mothers (p < 0.001), and pups from GH-treated mothers continued to grow faster (p < 0.001) than pups from control mothers throughout the postnatal growth period through to weaning. In normal Wistar rats, administration of a potent antiserum to somatostatin to pregnant rats increased the mean birth weight of the offspring (p < 0.01). Administration of GH to the mothers also significantly increased birth weight (p < 0.05), but administration of antiserum to rat GH resulted in a significant retardation of both fetal and placental growth (p < 0.001). These data suggest that maternal GH status is significantly involved in the growth of the fetal rat.

Neuroendocrine regulation of growth hormone secretion in sheep. VI. Intracerebroventricular administration of galanin.

The effects of intracerebroventricular (i.c.v.) administration of galanin on plasma growth hormone (GH) concentrations and its possible mechanism of action have been examined in sheep. Galanin administration icv resulted in a dose-dependent increase in plasma GH concentrations (p < 0.01). Concurrent administration of somatostatin (0.5 microgram kg-1 i.v. over 30 min) caused a delay in the GH response to galanin, but did not inhibit the GH release. These data show that galanin can stimulate GH release in sheep, but the mechanism through which this effect is mediated remains unclear.

Design and synthesis of an orally active macrocyclic neutral endopeptidase 24.11 inhibitor.

A potent macrocyclic inhibitor of neutral endopeptidase (NEP) 24.11 was designed using a computer model of the active site of thermolysin. This 10-membered ring lactam represents a general mimic for any hydrophobic dipeptide in which the two amino acid side chains bind to an enzyme in a contiguous orientation. The parent 10-membered ring lactam was synthesized and exhibited excellent potency as an NEP 24.11 inhibitor (IC50 = 3 nM). In order to improve oral bioavailability, various functionality was attached to the macrocycle. These modifications lead to CGS 25155, an orally active NEP 24.11 inhibitor that slows down the degradation of the cardiac hormone atrial natriuretic factor, producing a lowering of blood pressure in the DOCA-salt rat model of hypertension.

Heterocyclic lactam derivatives as dual angiotensin converting enzyme and neutral endopeptidase 24.11 inhibitors.

A series of 13- and 14-membered ring lactam derivatives 9a,b, 10, 11, and 12a-c was prepared from L-cysteine. Compounds 9a,b and 12a,b were tested in vitro for inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin converting enzyme (ACE) inhibition. The structure-activity profile of the series is discussed. Compound 9b, a 13-membered ring macrocyclic lactam, had an NEP IC50 of 18 nM and an ACEIC50 of 12 nM in vitro and showed dual plasma inhibition after intravenous or oral administration.

Evidence of a role for growth hormone, but not for insulin-like growth factors-I or -II in the growth of the neonatal rat.

Neonatal rats were injected with antiserum raised against either insulin-like growth factor (IGF)-I, IGF-II, rat growth hormone (rGH) or somatostatin (SRIF) on each of days 2-5 of life: controls received normal sheep immunoglobulin. Plasma levels of rGH and IGF-I were measured by radioimmunoassay and growth rates recorded. Neonatal administration of anti-rGH resulted in the suppression of plasma IGF-I levels at day 21 and of body weight gain compared with control animals from day 5 of age; relative growth velocity continued to diverge in the absence of any further treatment. Immunoneutralization of IGF-I or of IGF-II had no effect on growth rates of rats at any time during the experiment and had no effect upon plasma rGH concentrations at day 21. However, at day 7, plasma rGH was lower in anti-IGF-I-treated rats than in controls; in contrast, plasma rGH in anti-IGF-II-treated animals at day 7 was higher than in controls. Plasma levels of IGF-I at 49 days of age were similar regardless of the neonatal immunization treatment received. Anti-SRIF treatment of neonatal rats was associated with elevated rGH levels, but no significant stimulation of growth. These results indicated that growth hormone, but not circulating IGF-I or IGF-II are essential for normal growth in the neonatal rat.

A role for cGMP during tetanus toxin blockade of acetylcholine release in the rat pheochromocytoma (PC12) cell line.

In order to identify the specific molecular mechanisms involved in neurosecretion, we investigated the mechanism of action of tetanus toxin, a potent presynaptic neurotoxin, in the rat adrenal pheochromocytoma PC12 cell line. It has recently been reported that tetanus toxin is a potent inhibitor of the release of depolarization-evoked 3H-acetylcholine (ACh) from nerve growth factor-differentiated PC12 cells (Sandberg et al., 1989a). In PC12 cells, as in many neural tissue preparations, cGMP accumulation in intact cells increased 6- to 17-fold when stimulated with veratridine (200 microM), carbachol (1 mM), Ba2+ (2 mM), or K+ (30 mM). Preincubation of the cells with tetanus toxin inhibits this accumulation by greater than 95%. The toxin dose-inhibition curves for 3H-ACh release and cGMP accumulation are similar, with half-maximal doses of tetanus toxin seen at approximately 5 nM. The time courses for the development of the effects of tetanus on 3H-ACh release and on cGMP accumulation were also similar. Protocols which elevated intracellular cGMP levels reversed the action of the toxin. For example, evoked ACh release was restored in intoxicated PC12 cells by a 15 min exposure to 100 microM 8-bromo-cGMP. The half-maximal dose was observed at 50 microM nucleotide. Examination of the nucleotide specificity revealed that only cyclic guanine analogs were effective in reversing the effects of tetanus toxin. These results suggested that the inhibition of depolarization-evoked cGMP accumulation is causally related to the action of tetanus toxin on neurosecretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the intoxication pathway of tetanus toxin in the rat pheochromocytoma (PC12) cell line. Binding, internalization, and inhibition of acetylcholine release.

Tetanus toxin was found to be a potent inhibitor of neurosecretion in the rat pheochromocytoma cell line PC12, a system in which biochemical and functional studies could be performed in parallel. Incubation of the cells with 10 nM tetanus toxin (3 h) led to an inhibition of acetylcholine release by 75-80% when evoked by 200 microM veratridine, 1 mM carbachol, or 2 mM Ba2+. The main characteristics of the inhibition process are: 1) the toxin is very potent, with threshold doses of 10 pM; 2) the action of toxin is blocked at low temperature (0 degrees C) and by antitoxin; 3) the effects are dose- and time-dependent; 4) a concentration-dependent lag phase precedes the onset of the inhibitory effects. Thus the PC12 cultures are a valid system for studies on the underlying molecular process in tetanus action. This system was exploited by the use of long term incubation studies to examine the processes responsible for the lag phase. When cells were incubated with 0.1 nM 125I-tetanus toxin, cell-associated toxin reached a plateau of 16 fmol of toxin/mg of protein, yet the toxic effects did not appear until 12 h. Further, PC12 cells were found to rapidly internalize tetanus toxin, with a half-life of 1-2 min, once it was bound to the surface of the cells. Thus, the lag phase results from steps that occur in the intracellular compartment after internalization. An important discovery was that the differentiation state of the PC12 cells was a critical factor in determining sensitivity to tetanus toxin. Cells that were cultured with nerve growth factor for 8-12 days were very sensitive to toxin. In contrast, acetylcholine release from nondifferentiated, autodifferentiated, or dexamethasone-treated cultures was insensitive to tetanus toxin. Since differential expression of high affinity tetanus toxin receptors cannot explain these results, it is concluded that PC12 cells are capable of expressing different forms of excitation-secretion coupling mechanisms. Tetanus toxin should prove a valuable probe to further distinguish these processes.

Plasma concentrations of transsulfuration pathway products during nasoenteral and intravenous hyperalimentation of malnourished patients.

We have monitored the plasma concentrations of products of the transsulfuration pathway in 11 undernourished noncirrhotic patients, and in 10 cachectic cirrhotic subjects, before and during nasoenteral nutrition with Vivonex (Norwich-Eaton Pharmaceuticals, Norwich, NY) or total parenteral nutrition (TPN) with FreAmine III (American McGaw, Irvine, CA). In the cirrhotic cases eating a mixed diet, levels of taurine, cysteine, plasma glutathione, and free choline were subnormal. During nasoenteral hyperalimentation, methionine was elevated while cysteine, glutathione, and free choline levels remained depressed. During TPN, levels of taurine, cysteine, protein-bound cysteine, glutathione, free choline, and phosphatidyl choline were depressed and methionine was elevated. In the noncirrhotic cases eating a mixed diet, only the free choline concentration was low. During nasoenteral hyperalimentation, the plasma levels of both free choline and total carnitine were depressed. During TPN, plasma levels of cystine, protein-bound cysteine, total carnitine, free choline, and phosphatidyl choline were subnormal. These data suggest that biosynthesis of several products of the transsulfuration pathway may be inadequate in both cirrhotic and noncirrhotic patients during TPN with FreAmine III.

Effects of opioid peptides and opiate alkaloids on insulin secretion in the rabbit.

In pentobarbital-anesthetized rabbits, iv injection of 9 nmol (31 micrograms) human beta-endorphin (beta h-endorphin)/kg BW caused a significant (P less than 0.05) increase in serum glucose and a significant decline in serum insulin during the subsequent 60 min. When 9 nmol/kg BW beta h-endorphin were injected simultaneously with 0.7 g glucose/kg BW, the clearance of serum glucose and the expected glucose-stimulated rise in serum insulin were both inhibited. The threshold dose for the insulinopenic effect of beta h-endorphin in the anesthetized, glucose-loaded rabbit was 0.09 nmol/kg BW. Threshold doses/kg BW were determined for six structurally related peptides found to possess insulinopenic activity: camel beta-endorphin, 0.09 nmol; N-arg-beta h-endorphin, 0.09 nmol; D-ala2-beta h-endorphin, 0.09 nmol; leu5-beta h-endorphin, 0.09 nmol; met-(O)5-beta h-endorphin, 0.9 nmol; and beta h-endorphin1-27, 0.9 nmol. Threshold dose/kg BW for somatostatin was 9 nmol. The following compounds were inactive at 9 nmol/kg BW: N-acetyl-beta h-endorphin; N-acetyl-arg-beta h-endorphin; beta h-endorphin2-31; beta h-endorphin6-31; beta h-endorphin(((1-5 + 6-31))); beta h-endorphin1-18 (gamma-endorphin); beta h-endorphin1-17 (alpha-endorphin); beta h-endorphin1-5 (met-enkephalin); leu5-beta h-endorphin (leu-enkephalin); met-NH2(5)-beta h-endorphin1-5 (met-enkephalinamide); D-ala2-leu5-beta h-endorphin1-5; D-ala2-N-me-phe4, met-(O)5-ol-beta h-endorphin1-5; and D-ala2-D-leu5-beta h-endorphin1-5. Ninety nmoles per kg BW of naloxone did not alter the insulinopenic effect of 0.9 nmol/kg BW beta h-endorphin. As little as 2.9 X 10(-10) molar beta h-endorphin inhibited glucose-stimulated release of insulin by rabbit pancreas slices in vitro. The capacities of the peptides and alkaloids to inhibit insulin secretion in vitro followed the same general order as the in vivo insulinopenic capacities. Naloxone at 2.9 X 10(-6) M did not reduce the antisecretagogue effect of 2.9 X 10(-8) M beta h-endorphin. These findings, when compared with previously described structure-activity relationships for opioid receptors, indicate the presence of a novel receptor for beta-endorphin in rabbit pancreas, the activation of which inhibits glucose-stimulated secretion of insulin.