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Time-dependent Hartree-Fock calculations have been performed for fusion reactions of He-4 + He-4 -> Be*-8, followed by He-4 + Be*-8 . Depending on the orientation of the initial state, a linear chain vibrational state or a triangular vibration is found in 12C, with transitions between these states observed. The vibrations of the linear chain state and the triangular state occur at ~9 and 4 MeV respectively.

The objectives of this study were to evaluate the effect of reproductive protocols and reproductive tract score on reproductive performance of dairy heifers and economic outcomes of breeding programs. Holstein heifers (n = 534), 13 ± 1 mo of age, were randomly assigned to 1 of 4 reproductive protocols. On the day of enrollment (d 0), heifers were palpated per rectum and received a score according to the maturity of their reproductive tract (1 = prepubertal; 2 = peripubertal; and 3 = pubertal). Estrous detection-control heifers (CON, n = 146) received no treatment and were inseminated on detection of estrus for 28 d. Prostaglandin F2α-treated heifers (PGED, n = 137) received 1 injection of PGF2α on d 0 and were inseminated on detection of estrus; heifers not inseminated by d 14 received a second injection of PGF2α and were observed for estrus and artificial insemination (AI) for an additional 14 d. Heifers enrolled in the estrous detection-timed AI (EDTAI, n = 140) treatment received a controlled internal drug-release (CIDR) insert on d 0, and 7 d later, the CIDR was removed and all heifers received an injection of PGF2α, heifers received AI on detection of estrus, and those not inseminated by 72 h after PGF2α received an injection of GnRH concurrent with AI. Heifers in the GnRH-timed AI (GTAI, n = 111) treatment received 1 injection of GnRH on d 0, on d 6 heifers received a CIDR insert and injections of GnRH and PGF2α, on d 13 the CIDR was removed and heifers received an injection of PGF2α, and 48 h later all heifers received an injection of GnRH and AI. Pregnancy was diagnosed at 32 ± 3 and 62 ± 3 d after AI. Cost of reproductive protocols and their economic outcomes were calculated for a 28 d period beginning at enrollment. Heifers in the PGED treatment were inseminated at a faster rate than CON heifers. A smaller proportion of prepubertal and peripubertal heifers were inseminated within 14 d of enrollment compared with pubertal heifers. Pregnancy per AI of CON and PGED heifers was greater compared with EDTAI and GTAI heifers. Proportion of GTAI heifers pregnant at the end of the 28-d breeding program was or tended to be smaller compared with PGED and CON heifers, respectively. Heifers in the CON and PGED treatments had the smallest cost per pregnancy followed by heifers in the EDTAI and GTAI treatments, respectively. When different scenarios were evaluated, however, the mean cost per pregnancy was smallest for PGED heifers. Cost per pregnancy generated was greatest for prepubertal heifers, whereas pubertal heifers had the smallest cost per pregnancy generated. Treatment of dairy heifers with PGF2α every 14 d until insemination and pregnancy results in the best economic outcomes, and screening heifers according to RTS may prove beneficial to identify heifers that may not be pubertal and would have compromised reproductive and economic performance in a breeding program. [ABSTRACT FROM AUTHOR]

The objectives were to evaluate the effect of synchronization protocols on follicular development and estradiol 17-β (E2) and progesterone (P4) concentrations in dairy heifers. In experiment 1, 36 heifers were assigned to 1 of 6 synchronization protocols in a 3 x 2 factorial design: presynchronization with GnRH on study d -6 or -9 [study d 0 = initiation of the Cosynch + CIDR (controlled internal drug releasing insert containing P4) protocol] or no presynchronization (control) and one injection of PGF22α or not on study d 0. In experiment 2, 126 heifers were assigned to 1 of 4 synchronization protocols in a 2 x 2 factorial arrangement: presynchronization or not with GnRH on study d -6 and injection of PGF22α or not on study d 0. In experiments 1 and 2, all heifers received a modified Cosynch protocol with CIDR for 7 d starting on study d 0. After the PGF22α of the Cosynch and removal of the CIDR, heifers were detected in estrus and inseminated. Those not inseminated by study d 10 received an injection of GnRH and were timed-inseminated. Ovaries were scanned by ultrasound on d 0, 2, and 5, daily from d 7 to 14, and on d 16. Blood samples collected on d 0, 2, 7, 9, and 16 were analyzed for P4, and the blood sample collected on d 9 was analyzed for E2. Pregnancy was diagnosed at 28 and 40 ± 3 d after artificial insemination. In experiment 1, there was a tendency for the presynchronization protocol to affect the proportion of heifers ovulating in response to the first GnRH injection of the Cosynch + CIDR protocol. In experiment 2, a greater proportion of presynchronized heifers ovulated in response to the first GnRH injection. Although heifers receiving PGF22α had larger ovulatory follicles on d 7 and before ovulation and shorter intervals to estrus and ovulation, these heifers tended to have decreased concentrations of E2 during proestrus. Presynchronization of dairy heifers with GnRH increased ovulation in response to the first GnRH injection, and treatment of heifers with PGF2α at initiation of the Cosynch + CIDR protocol increased the size of the ovulatory follicle and reduced the intervals to estrus and ovulation. [ABSTRACT FROM AUTHOR]

The objective of the present study was to evaluate different synchronization protocols for Holstein heifers. Holstein heifers (n = 236), between 13±1 mo of age, were assigned to one of four synchronization protocols. Heifers in the CON group received no treatment and were AI upon detection of estrus; heifers in the PGF group received one injection of PGF2a on study d 0 and were AI upon detection of estrus, and those not inseminated by study d 14 received a second injection of PGF2a; heifers in the CIDR group received a CIDR insert on study d 0 for 7 d and an injection of PGF2a at CIDR removal and were AI upon detection of estrus from study d 7 to 10, and those not inseminated by 72 h after PGF received an injection of GnRH and were AI at fixed time; heifers in the TAI group received one injection of GnRH on study d -6, a CIDR insert, and an injection of GnRH and PGF2a on d 0, on study d 7 CIDR was removed and heifers received an injection of PGF2a, and 48 h later heifers received an injection of GnRH and were AI at fixed time. Heifers from CON and PGF groups not inseminated by study d 28 were right censored. Pregnancy was diagnosed at 29±3 and 62±3 d after AI. Data were analyzed using the LOGISTIC, LIFETEST, and GLM procedures of SAS. Treatment affected the interval from enrollment to AI (CON = 13.6±0.9, PGF = 9.6±1.1, CIDR = 10.3±0.1, TAI = 15.0±0.0 days; P = 0.007) and the proportion of heifers not inseminated by study d 28 (CON = 12.7, PGF = 15.9, CIDR = 0.0, TAI = 0.0%; P = 0.001), and tended to affect the interval from enrollment to conception (CON = 16.4±0.9, PGF = 14.5±1.2, CIDR = 10.6±0.7, TAI = 15.0±0.0 days; P = 0.06). Treatment tended to affect conception rate (CON = 66.1, PGF = 69.8, CIDR = 56.2, TAI = 44.8%; P = 0.10), but did not affect pregnancy rate after a 28 d breeding period (P = 0.63). Heifers from the CIDR group tended to become pregnant at a faster rate. [ABSTRACT FROM AUTHOR]

The objective of the present study was to evaluate the correlation between reproductive status and steady-state levels of Mx2 mRNA in peripheral blood mononuclear cells of dairy heifers. Holstein heifers (n = 257), 13 ± 1 mo of age, were assigned to be inseminated (BRED; n = 209) or not (NON-BRED; n = 48) after the completion of a synchronization protocol. All heifers received a CIDR insert for 7 d and at CIDR removal heifers received one injection of PGF2a (study d 0). Heifers from the BRED group were inseminated upon detection of estrus from study d 0 to 3, and those not inseminated received a GnRH injection concomitant with fixed time AI 72 h after PGF2a injection (study d 3). All heifers from the NON-BRED group received a GnRH injection on study d 2 to induce ovulation and were not inseminated. Blood samples collected on study d 2 and 20 were used to determine steady-state levels of Mx2 mRNA using quantitative real-time PCR, and fold increase in Mx2 mRNA levels from study d 2 to 20 was calculated. Pregnancy was diagnosed at 30 and 65 d after AI. Data were analyzed using GLM procedure of SAS. Pregnancy rate at 30 d and 65 d after AI were 49.6% and 43.7%, respectively, and pregnancy loss between 30 d and 65 d after AI was 11.9%. Heifers diagnosed as pregnant on d 30 (NON-BRED = 1.61 ± 0.41, BRED/Non-pregnant = 2.03 ± 0.34, BRED/Pregnant = 2.98 ± 0.34 fold) and 65 after AI (NON-BRED = 1.61 ± 0.41, BRED/Non-pregnant = 2.12 ± 0.32, BRED/Pregnant = 2.99 ± 0.36 fold) had greatest (P = 0.03) Mx2 mRNA fold change between study d 2 and 20. There was no difference (P = 0.94) in Mx2 mRNA fold increase between pregnant heifers that experienced or did not experience fetal loss between 30 and 65 d after AI. The range of Mx2 mRNA fold increase was as follow: NON-BRED = 0.05-13.02; BRED/Non-pregnant = 0.05-13.08; BRED/Pregnant = 0.15-14.32; and Bred/Aborted = 0.19-6.73. The present study indicates that increased levels of Mx2 mRNA in peripheral blood mononuclear cells are related to pregnancy at 30 and 60 d after AI in dairy heifers. [ABSTRACT FROM AUTHOR]

The objective of the present study was to evaluate the effect of synchronization protocols on follicular development of dairy heifers. Holstein heifers (n = 151), 13 mo of age, were assigned to one of four synchronization protocols in a 2 x 2 factorial arrangement, presynchronization (PRES) or no presynchronization (NPRES) with GnRH on study d -6 (study d 0 = initiation of the Co-Synch) and an injection of PGF2a (PGF) or no injection of PGF2a (NPGF) on study d 0. This resulted in 4 treatments (NPRES and NPGF; PRES and NPGF; NPRES and PGF; PRES and PGF). On d 0, all heifers received the Co-Synch protocol with a CIDR insert for 7 d. After the PGF2a of the Co-Synch, heifers detected in estrus were AI, and those not AI by d 10 were timed AI and received the final injection of GNRH of the Co-Synch. Ovaries were scanned by ultrasound on d 0, 2, 5, and daily from d 7 to 14. Blood samples collected on d 0, 2, 7, 9, and 16 were analyzed for P4. Pregnancy was diagnosed at 29 d after AI. Data was analyzed using GLM and CHISQ procedures of SAS. Greater proportion of heifers presynchronized ovulated in response to the GnRH injection given on d 0 (NPRES = 30.7 vs. PRES = 54.0%, P = 0.004). Presynchronization did not affect P4 concentration on d 0 (P = 0.80), but tended to affect it on d 2 (NPRES = 4.5 ± 0.3 vs. PRES = 5.4 ± 0.3 ng/mL; P = 0.06). Treatment with PGF2a on d 0 affected (P < 0.001) P4 concentration on d 2 (NPGF = 6.4 ± 0.3 vs. PGF = 3.5 ± 0.3 ng/mL) and 7 (NPGF = 2.7 ± 0.2 vs. PGF = 1.4 ± 0.2 ng/mL). Size of the ovulatory follicle on d 7 was not affected (P = 0.50) by pre-synchronization treatment, but it was affected by treatment with PGF2a on d 0 (NPGF = 13.5 ± 0.3 vs. PGF = 15.0 ± 0.3 mm; P = 0.001). Treatment with PGF2a on d 0 affected interval from CIDR removal to ovulation (NPGF = 3.7 ± 0.1 vs. PGF = 3.4 ± 0.1 d; P = 0.004). Presynchronization treatment did not affect P4 concentration on d 16 (P = 0.60), but heifers that received a PGF2a injection on d 0 had greater P4 concentration on d 16 (NPGF = 2.8 ± 0.2 vs. PGF = 3.5 ± 0.2 ng/mL; P = 0.002). Pregnancy rate was not affected by presynchronization treatment (P = 0.85) or treatment with PGF2a on d 0 (P = 0.99). [ABSTRACT FROM AUTHOR]

Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. FGR pregnancies are often associated with histological evidence of placental vascular thrombosis. The proteoglycans are important components and regulators of vascular homeostasis. Previous studies from our laboratory highlighted mRNA and protein expression differences in placental proteoglycan decorin (DCN), within a clinically well-characterised cohort of third-trimester idiopathic FGR compared with gestation-matched uncomplicated control pregnancies. We also showed that decorin contributes to abnormal angiogenesis and increased thrombin generation in vitro. These observations suggest that DCN gene expression may contribute to the etiology of FGR. Small for gestational age (SGA) is frequently used as a proxy for FGR and is defined as a birth weight below the 10th percentile of a birth weight curve. We therefore made use of a unique resource of first trimester tissues obtained via chorionic villus sampling during the first trimester to investigate the temporal relationship between altered DCN expression and any subsequent development of SGA. We hypothesized that placental DCN expression is decreased early in gestation in SGA pregnancies. Surplus chorionic villus specimens from 15 women subsequently diagnosed with FGR and 50 from women with uncomplicated pregnancies were collected. DCN mRNA and DCN protein were determined using real-time PCR and immunoblotting, respectively. Both DCN mRNA and protein were significantly decreased in placentae from first-trimester SGA-pregnancies compared with controls (p < 0.05). This is the first study to report a temporal relationship between altered placental DCN expression and subsequent development of SGA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Calreticulin is a ubiquitously expressed protein that was detected in the circulation and is significantly increased in maternal blood during human pregnancy compared to the non-pregnant state. Calreticulin is further increased in the plasma of women with the pregnancy-related disorder pre-eclampsia compared to normotensive pregnancy. The aims of this study were to compare calreticulin in human pregnancy with calreticulin in rat pregnancy, and to compare calreticulin during fetal growth restriction with normal control pregnancies., Methods: Women were recruited who either had normal pregnancies or had pregnancies complicated with fetal growth restriction; maternal blood samples and placentas were collected. Blood was also taken from women who were not-pregnant. Growth restriction was induced in pregnant rats by uterine vessel ligation; blood and placental samples were collected. Blood was also taken from non-pregnant rats. Western blot was used to quantify the placental expression of calreticulin and the concentrations of calreticulin in plasma., Results: Although calreticulin was significantly increased in maternal plasma during human pregnancy compared to the non-pregnant state; it did not increase in plasma during rat pregnancy. These results suggest that there may be differences in the role of extracellular calreticulin in human compared to rat pregnancy. Calreticulin was not significantly altered in either placental extracts or maternal plasma in both the human and rat pregnancies complicated by fetal growth restriction compared to gestational matched control pregnancies., Conclusion: This study found that there was no change in calreticulin during human pregnancy complicated with fetal growth restriction or when growth restriction is induced in rats., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Chloride intracellular channel (CLIC) proteins constitute a subgroup of the glutathione-S-transferase (GSTs) superfamily. In humans, the CLIC family of proteins consists of six members, designated CLIC 1-6, which have a conserved C-terminal 240 residue module and one major transmembrane domain. CLIC proteins regulate fundamental cellular processes including regulation of chloride ion concentration, stabilization of cell membrane potential, trans-epithelial transport, regulation of cell volume and stimulation of apoptotic processes in response to cellular stress. Previously, we described the expression profile of a member of the CLIC family of proteins, CLIC3, in human placentae and fetal membranes. In the current study, we determined CLIC3 expression in placentae from pregnancies complicated with either fetal growth restriction (FGR, n=19), pre-eclampsia (PE, n=16) or both FGR and PE combined (n=12) compared to gestation-matched controls (n=13) using real-time PCR and a CLIC3 specific immunoassay. Significantly increased CLIC3 mRNA and protein were detected in placental extracts from pregnancies with FGR, PE and PE with FGR compared to controls. Our results suggest that increased expression of CLIC3 may play a role in abnormal placental function associated with the human pregnancy disorders FGR and PE., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Calreticulin is a calcium binding, endoplasmic reticulum resident protein best known for its roles in intracellular calcium homeostasis and the quality control processes of the endoplasmic reticulum. There is evidence for a range of activities for calreticulin outside the endoplasmic reticulum, including in the cytosol, on the surface of different cells types and in the extracellular matrix. Recent evidence indicates that human pregnancy is a condition of elevated circulating calreticulin. Calreticulin was increased in the plasma of women throughout pregnancy compared to the non-pregnant state. Calreticulin was also further increased during the hypertensive disorder of human pregnancy, pre-eclampsia. To clarify the roles of circulating calreticulin in pregnancy and pre-eclampsia, the aim of this study was to determine the effects of exogenous calreticulin on two cell types that are relevant to normal human pregnancy and to pre-eclampsia. Human primary myometrial microvascular endothelial cells (UtMVEC-Myo) and the human trophoblast cell line, HTR8/SVneo, were cultured with exogenous calreticulin at concentrations (2 μg/ml and 5 μg/ml) comparable to that measured in maternal blood. The higher concentration of calreticulin significantly increased the migration of the UtMVEC-Myo cells, but significantly reduced the migration of the HTR8/SVneo cells. In the presence of only FGF, FBS and antibiotics calreticulin at 5 μg/ml significantly reduced the number of UtMVEC-Myo cells during in vitro culture for 120 h. These results demonstrate that exogenous calreticulin can alter both HTR8/SVneo and UtMVEC-Myo cell functions in vitro at a (patho-) physiologically relevant concentration. Increased calreticulin may also contribute to altered functions of both cell types during pre-eclampsia., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pre-eclampsia is a disorder of human pregnancy that involves pregnancy-induced maternal hypertension and proteinuria. Evidence indicates that pre-eclampsia involves widespread activation of maternal endothelial cells. Calreticulin is a ubiquitously expressed, multi-functional protein that has been shown to have both pro- and anti-inflammatory effects on cultured endothelial cells in vitro and in whole animals. In order to clarify the role of this protein in normal human pregnancy and in pre-eclampsia, this study has measured expression of calreticulin in maternal blood and in placenta in patients with pre-eclampsia and in control pregnancies. There was a significant increase (approximately 5-fold) in calreticulin in plasma in term pregnant women compared with women who were not pregnant. There was no difference, however, in calreticulin in plasma from women who were sampled at first trimester, second trimester and at term. In addition, there was a significant increase (approximately 50%) in calreticulin in plasma from pre-eclamptic women compared to controls. Calreticulin mRNA and protein expression in placenta were not changed between pre-eclampsia and control pregnancies. These novel results indicate that calreticulin is increased in peripheral maternal blood early in pregnancy and remains elevated throughout normal gestation and that there is a further increase in calreticulin in pre-eclampsia.

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.

The aim of this study was to characterize the expression of the novel glucose transporter GLUT12 in the fetal membranes of the human placenta. RT-PCR and Western blotting of extracts of amnion and choriodecidua from four normal term placentas identified GLUT12 mRNA and protein expression. In all four samples the signals for GLUT12 were markedly stronger in the choriodecidua than in the amnion, whereas the signals for GLUT1, a glucose transporter know to be expressed in fetal membranes, were similar for the two tissues. In further studies, paraffin sections of fetal membranes were analyzed by immunohistochemistry with GLUT12 and GLUT1-specific polyclonal antibodies. GLUT12 immunoreactivity was localized predominantly to the trophoblast cells in the chorion and to a lesser extent to decidual cells and to epithelial and fibroblast cells of the amnion. GLUT1 was localized to chorionic trophoblast cells and amniotic epithelial and fibroblast cells. GLUT12 expression was predominantly cytoplasmic, whereas GLUT1 was associated with the membrane of the cells. These results show that GLUT12 is expressed in cells of human fetal membranes and suggest that GLUT12 may play a role in the facilitation of glucose transport into these cells.

The aim of this study was to characterize the expression of a novel glucose transporter protein GLUT12 in human placenta. GLUT12 mRNA expression was identified by RT-PCR in extracts from five normal term placentae and in extracts from cultured cells of the JAR, JEG-3 and HTR-8Svneo cell lines. In further studies, paraffin sections of first trimester tissue from chorionic villus sampling and term tissue obtained after delivery were analysed by immunohistology with a GLUT12 specific polyclonal antibody. GLUT12 immunoreactivity was expressed predominantly in the syncytiotrophoblast and in extra-villous trophoblast cells in first trimester tissues at 10, 11 and 12 weeks' gestation. In term tissue, however, GLUT12 staining was not detected in syncytiotrophoblast and was found predominantly in villous vascular smooth muscle cells and villous stromal cells. These results suggest that there is a dynamic spatial and temporal expression pattern for the novel glucose transporter GLUT12 in human placenta.

These studies have established the presence of two major classes of high affinity growth hormone binding proteins in pregnant rat serum, designated GHBPa and GHBPb, with apparent native Mr of 257 K and 98 K respectively. GHBPa, which has not been identified previously, exhibits a binding affinity (2-5 nM(-1)) that is up to 20-fold higher than GHBPb (0.2-0.8 nM(-1)) and is the least abundant form, being approximately 15-20% of total serum GH-binding capacity. Western immunoblot analysis revealed that each GHBP is composed of several immunoreactive proteins which were reactive with carboxy-terminal (RB1615) and/or N-terminal (MAb263) domain antibodies, suggesting the presence of GHBPs with and without the hydrophilic tail. Of importance is that GHBPa exhibited significantly higher Mr (78-182 K, +DTT) than that predicted by GHBP cloning, suggesting that they may be covalently bound to other non-GH-binding proteins or may be distinct entities. GHBPb, on the other hand, was composed of smaller Mr (43/48 K, +DTT) "hydrophilic" tail-containing proteins, some of which were disulphide linked to a larger complex of approximately 110 K. These novel findings challenge the current view of the mechanism for generation of the rat serum GHBP and raise the intriguing possibility that the two classes of GHBP may play distinct and important roles in GH physiology., (Copyright 2000 Harcourt Publishers Ltd.)

The solution structure of human erythropoietin (EPO) has been determined by nuclear magnetic resonance spectroscopy and the overall topology of the protein is revealed as a novel combination of features taken from both the long-chain and short-chain families of hematopoietic growth factors. Using the structure and data from mutagenesis studies we have elucidated the key physiochemical properties defining each of the two receptor binding sites on the EPO protein. A comparison of the NMR structure of the free EPO ligand to the receptor bound form, determined by X-ray crystallography, reveals conformational changes that may accompany receptor binding.

Previous workers have suggested that guinea pig serum does not contain a GH-binding protein (GHBP) or that it is defective. The current studies, however, have identified and characterized the presence of GH-binding activity in guinea pig serum using gel chromatography to separate bound and free hormone. The detection of GH-binding activity is critically reliant on the type of radioligand used to measure binding. Clear identification of GH-binding activity was demonstrated with [125I]ovine GH (oGH), but specific binding could not be measured with [125I]human GH. The novel specificity was also shared by guinea pig liver membrane GH receptor (GHR) and cytosol GHBP, suggesting structural similarity in the GH-binding domain between the GHR and soluble GHBPs. The binding of oGH was dependent on serum concentration (5 microl serum produced 16.03 +/- 0.5% specific binding; mean +/- SEM; n = 11) and incubation time (equilibrium was reached by approximately 6 h at 21 C) and was completely reversible (t(1/2), approximately 2 h). Scatchard analysis revealed linear plots with an affinity constant (Ka) of 0.59 +/- 0.09 x 10(9) M(-1) and a capacity of 23,181 +/- 4,474 fmol/ml serum. Similar association constants were obtained for liver membrane GHR (0.79 +/- 0.22 x 10(9) M(-1)) and cytosol GHBPs (0.99 +/- 0.15 x 10(9) M(-1)), but the capacity, when expressed as femtomoles per g tissue, was significantly increased (4-fold) in cytosol (4,303 +/- 505) over that in membranes (1,071 +/- 257). There was no sex difference in Ka or level of GHBP in guinea pig serum. Surprisingly, the level of GH-binding activity was very low to undetectable in pregnant guinea pig serum. Characterization of the native structure of guinea pig GHBPs has indicated the presence of several proteins that are structurally distinct. Although the distribution of GH-binding activity covered a large Mr range (approximately 70-350 kDa) the major form of the circulating GHBP identified by gel chromatography had an apparent native Mr of 150-170 kDa. Partially purified GHBP (approximate Mr, 170 kDa) was covalently cross-linked to [125I]oGH and subjected to nonreducing SDS-PAGE. Specific GHBP complexes of 158 and 85 kDa were detected, suggesting that the partially purified GHBP complex may be composed of a smaller GHBP associated noncovalently with a non-GH-binding protein. "Pore limit" native PAGE (cathodic and anodic) revealed the presence of specific GHBPs of 363, 158, 74, and 55 kDa, which cross-hybridized with the rat liver membrane GHR monoclonal antibody mAb 263 but not with the rat serum GHBP-specific mAb 4.3. Interestingly, although GH binding was undetectable in pregnant guinea pig serum, Western immunoblot analysis with mAb 263 demonstrated the presence of a major immunoreactive GHBP band of 105 kDa in addition to 158- and 55-kDa GHBPs. The data indicate that the GHBPs are immunologically related to the rat membrane GHR, but provide no evidence to support the presence of a hydrophilic tail sequence homologous to that in the rat GHBP. These studies have identified in guinea pig serum GHBPs that exhibit novel ligand specificity, structural heterogeneity, and an immunological relationship to the rat liver membrane GHR. The identification of serum GHBP and the novel ligand specificity, which is also expressed by the liver membrane GHR, argue against the view that the guinea pig has a defective GHBP.

We have modified the tryptophanase promoter (PtnaA) for use as a temperature-independent promoter for the production of recombinant proteins. Although any protein will have a temperature range in which its expression is optimal, we find the tryptophanase promoter functions at all physiologically relevant temperatures (20 degrees C to 42 degrees C). Induction at temperatures below 37 degrees C avoids eliciting the heat-shock response and may favor the production of protein in the soluble state. A short segment of the E. coli tnaA promoter containing the catabolite gene activator protein (CAP) binding site but no tryptophan-responsive elements was used to direct synthesis of various proteins. Conditions for high cell density fermentation and induction control were developed. Expression was induced by depletion of glucose and was maximal when an alternative nonrepressing carbon source was supplied. Expression of certain proteins was tightly controlled; however, pre-induction expression was observed with other reporter genes. The tnaC leader portion of the tnaA promoter was found to reduce pre-induction expression in the presence of glucose, although maximal expression was observed only in the absence of this region. The effect of temperature on expression of several recombinant proteins was investigated. Although some proteins were produced only in inclusion bodies as insoluble material, the production of one protein in soluble form was clearly temperature dependent.

Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular form(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR.

To determine whether arachidonic acid (AA) alters alveolar epithelial permeability, we studied the effect of both continuous intravenous and aerosolized AA on clearance of [99m]Tc-DTPA from lung to blood in rabbits. Although intravenous AA increased prostacyclin production and aerosolized AA decreased systemic blood pressure, neither continuous intravenous nor aerosolized AA augmented alveolar epithelial permeability.

The effect of elevated serum ovine GH (oGH) concentration on liver somatotrophic and lactogenic receptors was studied in transgenic mice expressing a metallothionein 1(MT)-oGH fusion gene. The mice belonged to three different pedigrees and were killed between 14 and 63 weeks of age. The levels of GH receptor (GH-R) and PRL receptor (PRL-R) determined by competitive binding assays were similar to those observed in late pregnant, nontransgenic mice. This observation was made for all transgenic mice expressing elevated serum oGH levels, irrespective of sex, final size, or age. Cross-linking studies revealed that binding occurred predominantly to a Mr 48,000 polypeptide with a small amount of binding to polypeptides of Mr 60,000, 70,000, and 100,000 in transgenic mice as well as in a late pregnant, nontransgenic mouse. Total cellular RNA was isolated from livers of transgenic and nontransgenic mice and analyzed on Northern blots using probes specific for GH-R and PRL-R. Results showed that the levels of messenger RNA for both GH-R and PRL-R were elevated in transgenic mice expressing high levels of serum oGH. Since levels of PRL in these mice were within the normal range, these results demonstrate that oGH is capable of inducing hepatic GH-R and PRL-R in vivo and that PRL is not required for the induction of its own receptor. These data also demonstrate, for the first time, the suitability of transgenic mice expressing a foreign GH for the study of the regulation of hepatic GH and PRL receptors.