Stimulation of the pituitary-adrenocortical axis by nerve growth factor.

Nerve growth factor (NGF) is a protein essential for the development and maintenance of the peripheral sympathetic nervous system, causing responsive neurones to increase in size and to extend neurites. Biochemically, the selective induction of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase key enzymes in catecholamine biosynthesis is one of its most characteristic effects. Both the morphological and biochemical effects are modulated by glucocorticoids, suggesting a close relationship between specific effects of NGF and hormone action. NGF has been shown to induce an increase in adrenal cyclic AMP in intact but not in hypophysectomised rats, and so we have looked directly at the effect of systemic administration of NGF on the hypothalamo-pituitary-adrenal axis. We report here that NGF induced an enhanced secretion of adrenocorticotropin (ACTH) and a prolonged increase in plasma glucocorticoid concentration after intravenous (i.v.) injection. Such effects could have important implications for the biological activity of NGF.     

Presence in brain of synenkephalin, a proenkephalin-immunoreactive protein which does not contain enkephalin

The primary sequence of adrenal proenkephalin has recently been reported by three groups who have isolated and sequenced the cDNA for this prohormone. Several intermediates in the processing of proenkephalin, containing from one to four copies of [Met] enkephalin, have been purified from the adrenal medulla. Although there is evidence that the proenkephalin is identical in the brain and the adrenal medulla, similar intermediates have not been isolated from brain. We report here the production of an antiserum directed against a purified enkephalin precursor derived from the amino terminus of adrenal proenkephalin which cross-reacts with an antigen in brain. The immunoreactive protein in brain does not contain the sequence of enkephalin, but shows a pattern of distribution in immunohistochemical studies parallel to that of the enkephalins. In extracts of bovine caudate-putamen, this antigen is present in a molar concentration approximately one-fifth of that of [Met] enkephalin. The results demonstrate that the antiserum recognizes antigenic determinants within the N-terminal 72 amino acid residues of adrenal proenkephalin and that the enkephalin precursor in brain is similar to that found in the adrenal medulla. Furthermore, the absence of the enkephalin sequence in the brain protein indicates that concentrations of the larger intermediates in the processing of proenkephalin are much lower in the brain than in the adrenal medulla.     

胎儿肾上腺的研究

本文观测了河南地区１９８例胎儿肾上腺。肾上腺重（ｙ）与体重、胎龄分别有很好的直线、指数曲线回归关系，回归方程为和；１５周胎肾上腺皮、髓质分明，皮质分化出球状、束状和网状三带，以后的发育中皮质所占比例变小，皮质中网状带渐薄，束状带渐厚。     

Circulating angiotensin II and adrenal receptors after nephrectomy

Mineralocorticoid secretion is predominantly controlled by the octapeptide angiotensin II, which exerts trophic actions on the adrenal glomerulosa and acute regulatory effects on aldosterone biosynthesis. The trophic actions include stimulation of angiotensin II receptors and enzymes of the aldosterone biosynthetic pathway, with corresponding enhancement of the aldosterone secretory capacity of the adrenal gland. The positive regulatory action of angiotensin II on its adrenal receptors occurs with elevations of the circulating peptide concentration within the physiological range and probably contributes to the increased sensitivity of the adrenal during sodium deficiency. In this action, angiotensin II differs from other hormones which decrease their target-cell receptors. However, the increase in adrenal angiotensin II receptors following nephrectomy has been interpreted as evidence for a tonic down-regulating effect of angiotensin II on its adrenal receptors. To clarify these conflicting views we evaluated the effects of nephrectomy on adrenal angiotensin II receptors in relation to blood angiotensin II and plasma electrolyte levels. We show here that hyperkalaemia contributes markedly to the post-nephrectomy increase in adrenal angiotensin II receptors, and that circulating angiotensin II levels persist for an unexpectedly long period after nephrectomy, presumably due to tissue generation of the octapeptide.     

Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins.

Corticotropin-releasing factor (CRF), is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRF concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRF probably originates from the placenta, which responds to the bioactive peptide and produces the peptide and its messenger RNA. Even though CRF concentrations in late gestational maternal plasma are similar to those in rat hypothalamic portal blood and to those that can stimulate release of adrenocorticotropic hormone (ACTH) in vitro, maternal plasma ACTH concentrations increase only slightly with advancing gestation and remain within the normal range. Several groups have now reported the existence of a CRF-binding protein in human plasma which inactivates CRF and which has been proposed to prevent inappropriate pituitary-adrenal stimulation in pregnancy. The binding protein was recently purified from human plasma. We have now isolated and partially sequenced the binding protein, allowing us to clone and characterize its complementary DNA from human liver and rat brain. Expression of the cDNAs for human and rat binding protein in COS7 cells showed that these proteins bind CRF with the same affinity as the native human protein. Both rat and human recombinant binding proteins inhibit CRF binding to a CRF antibody and inhibit CRF-induced ACTH release by pituitary cells in vitro.     

Evidence that the insulin secretagogue, beta-cell-tropin, is ACTH22-39

The pituitary neurointermediate lobe of genetically obese (ob/ob) mice contains a hormone which stimulates insulin release and which cross-reacts with a -COOH-terminal ACTH antiserum, suggesting that it is related to corticotropin-like intermediate lobe peptide (CLIP), the 18-39 fragment of ACTH. The hormone, which we have called beta-cell-tropin, has been shown to be present in the plasma of the ob/ob mouse and to potentiate glucose induced insulin secretion. We have now shown that ACTH22-39 prepared by tryptic digestion of human synthetic CLIP behaves similarly on Biogel chromatography and on reverse-phase HPLC to the naturally occurring beta-cell-tropin. Furthermore, beta-cell-tropin and ACTH22-39 have indistinguishable antigenic and insulin releasing properties.     

Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin

The stress-induced release of ACTH is believed to involve the activation of several humoral and neural pathways, including corticotropin-releasing factor (CRF), catecholamines and vasopressin. The essential role of CRF was supported by our observation that immunoneutralization of this releasing factor significantly lowers plasma ACTH levels of ether-stressed rats. However, the presence of a small but measurable residual ACTH secretion suggested the possible involvement of factors other than CRF in the stress response. We report here that pretreatment with a vasopressin antagonist decreases the plasma ACTH levels of ether-stressed rats in later (10-20 min), but not earlier (0-10 min), phases of ether stress. The ganglionic blocker chlorisondamine, inhibits ACTH release during both phases of the response to ether by 40-60% when used alone, and by 100% when administered with anti-CRF antibody. These results support a role of CRF, catecholamines and vasopressin in mediating ACTH release by ether stress.     

Production of gene-targeted sheep by nuclear transfer from cultured somatic cells

It is over a decade since the first demonstration that mouse embryonic stem cells could be used to transfer a predetermined genetic modification to a whole animal. The extension of this technique to other mammalian species, particularly livestock, might bring numerous biomedical benefits, for example, ablation of xenoreactive transplantation antigens, inactivation of genes responsible for neuropathogenic disease and precise placement of transgenes designed to produce proteins for human therapy. Gene targeting has not yet been achieved in mammals other than mice, however, because functional embryonic stem cells have not been derived. Nuclear transfer from cultured somatic cells provides an alternative means of cell-mediated transgenesis. Here we describe efficient and reproducible gene targeting in fetal fibroblasts to place a therapeutic transgene at the ovine alpha1(I) procollagen (COL1A1) locus and the production of live sheep by nuclear transfer.     

Functional corticotropin releasing factor receptors in the primate peripheral sympathetic nervous system

Corticotropin releasing factor (CRF) is a key hormone in the integrated response to stress, acting both as the major regulator of pituitary adrenocorticotropic hormone (ACTH) release and as a neuropeptide in the brain. The actions of CRF are mediated by specific plasma membrane receptors in the anterior pituitary gland and in discrete brain areas including the cerebral cortex and several regions related to the limbic system. In addition to the pituitary and central actions of CRF, systemic administration of the peptide in the rat, dog, monkey and man causes hypotension and tachycardia because of a decrease in peripheral vascular resistance. These observations, in conjunction with the finding of immunoreactive and bioactive CRF in peripheral tissues, suggest that the peptide is locally released in tissues to act as a neurotransmitter or paracrine hormone. As CRF is present in the adrenal medulla and the peptide is known to modulate the central activity of the autonomic nervous system, we investigated the possibility that CRF is involved in the regulation of the peripheral autonomic nervous system. Such an action of CRF is supported by our demonstration of specific CRF receptors in the monkey adrenal medulla and sympathetic ganglia. In the adrenal medulla, these receptors are coupled to adenylate cyclase and can stimulate the secretion of catecholamines and Met-enkephalin.     

Lactogenic receptor regulation in hormone-stimulated steroidogenic cells

Receptor sites for lactogenic hormones such as prolactin (PRL), human growth hormone (HGH), and placental lactogens, are widely distributed in mammalian tissues, including mammary glands, steroid-secreting cells of the adrenal, testis, and ovary, and target cells of steroid hormone action such as liver, prostrate, and kidney. Although the biological functions of lactogen binding sites remain uncertain, a relationship between prolactin and lipoprotein metabolism is implied by the occurrence of prolactin receptors in steroidogenic cells of the gonads and adrenal, and by the ability of prolactin to increase esterified cholesterol in the testis. Recently, loss of testicular prolactin receptors has been observed following elevation of circulating luteinising hormone (LH) concentrations by the gonadotropin releasing hormone (GnRH) and its agonist analogues. The hormone dependence of lactogen receptor sites in steroid-secreting cells was further analysed in rat testis, ovary, and adrenal glands after treatment with the respective trophic hormones, gonadotropin and ACTH. In each of these tissues, rapid and transient loss of lactogen receptors was observed after trophic hormone stimulation. These findings indicate that increased turnover of lactogen receptors is an important component of the target-cell response, and suggest that prolactin receptors might be involved in the transport of lipoprotein precursors for steroid biosynthesis.     

A haemoglobin switching activity modulates hereditary persistence of fetal haemoglobin

During human development there is a switch from fetal to adult haemoglobin formation, reflecting the differential expression of fetal (G gamma and A gamma) and adult (beta and delta) globin genes. Mutations that inhibit this switch produce variants of the syndrome of hereditary persistence of fetal haemoglobin (HPFH). Adult heterozygotes for these mutants produce 15-30% fetal haemoglobin (HbF) in their red cells. The general assumption is that the mutations result in a permanent switching on of gamma-globin genes. Here, however, we show that fetal globin expression can be turned off in cultures of HPFH cells by an uncharacterized factor in fetal sheep serum. This is the first demonstration that mutations affecting the developmental expression of globin genes can be modulated by exogenous factors. The findings raise the possibility that the phenotype of HPFH is not simply the direct result of mutations in or around globin genes but the consequence of the mutations on the interaction of globin genes with trans-acting regulatory factors.     

Cloned pigs produced by nuclear transfer from adult somatic cells

Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.     

Met-enkephalin circulates in human plasma

The physiological roles of Met-enkephalin and Leu-enkephalin are still unknown. They may act as neurotransmitters in the central and peripheral nervous systems. Met-enkephalin has been detected in several species in a variety of tissues including brain, spinal cord and gut using bioassays, opiate receptor assays and radioimmunoassays (RIA). It has also been detected in human gut immunocytochemically and in human brain and cerebrospinal fluid by opiate receptor assay and RIA. However, all reported assays show some degree of cross-reaction with Leu-enkephalin and unequivocal differentiation between the two enkephalins and the larger endorphins has not always been possible. Thus the existence of Met-enkephalin in human tissues and fluids remains in doubt. Using a highly specific RIA, we have now obtained evidence that Met-enkephalin-like material circulates in the plasma of normal subjects and may be secreted by the adrenal gland. Chromatographically the material exists in plasma mainly as the intact pentapeptide and not as the biologically inactive degradation product Gly-Gly-Phe-Met as would be expected from metabolic studies.     

Immunoreactive beta-endorphin in sheep ovary

Although the ovarian production of sex steroids is of obvious physiological importance, recent studies suggest that peptides such as oxytocin, relaxin and inhibin are also synthesized in the ovary. We report here the presence of immunoreactive (ir) beta-endorphin, ir-adrenocorticotropic hormone (ACTH) and presumptive high molecular weight forms of both in extracts of sheep ovary, consistent with ovarian production from a common precursor. Our findings suggest that beta-endorphin and ACTH are produced and secreted by the follicular cells, and that their production may be related to the oestrous cycle.     

Evidence for local stimulation of ACTH secretion by corticotropin-releasing factor in human placenta

The hypothalamic-pituitary-adrenocortical axis is activated in pregnancy and parturition. Levels of immunoreactive corticotrophin releasing factor (irCRF), immunoreactive adrenocorticotropic hormone (irACTH) and cortisol concentrations in maternal plasma are elevated throughout gestation, increase further during labour and fall precipitously after parturition. The placenta contains biologically active CRF and ACTH and it has been suggested that the placenta produces these peptides during pregnancy. Here we show that irCRF is located in the cytotrophoblast cells of placenta collected at term. Using a monolayer primary culture of human placental cells we have found that CRF stimulates secretion of peptides containing the ACTH sequence in the placenta in a dose-dependent manner, as it does in the pituitary. This effect is reversed by a CRF antagonist and is mimicked by dibutyryl cyclic AMP and forskolin. Glucocorticoids, which suppress the secretion of pituitary ACTH, were found to have no influence on release of irACTH by the placenta. Oxytocin and prostaglandins stimulate irACTH and irCRF secretion from cultured placental cells and the irACTH-releasing activity of two prostaglandins is partially reversed by a CRF antagonist. Thus CRF may be involved in the paracrine regulation of placental irACTH secretion.     

针刺对焦虑障碍大鼠作用的部分机制研究

目的：分析针刺对焦虑障碍大鼠肾上腺心房利钠肽、C型利钠肽水平和血浆皮质酮含量的影响,探讨针刺治疗焦虑障碍的部分机制.方法选取SPF级SD大鼠随机分为空白对照组、模型组、针刺组.模型组、针刺组大鼠建立慢性情绪应激刺激焦虑模型,针刺组同时针刺“内关”、“神门”,每2 d针刺1次,45 min/次,共15 d.空白对照组和模型组用相同方法固定,不做任何干预.使用高架十字迷宫观察大鼠行为学变化,计算进入开臂次数( OE)和停留时间( OT)分别占进入总次数和停留时间的百分比;ELISA法检测大鼠血浆皮质酮水平;免疫组化S-P法检测大鼠肾上腺心房利钠肽、C型利钠肽水平.结果针刺组大鼠OT值明显高于模型组,差异具有统计学意义(P<0.05),与空白对照组之间差异无统计学意义(P>0.05);3组大鼠OE值之间差异均无统计学意义(P>0.05).模型组大鼠浆皮质酮含量明显高于空白对照组,差异具有统计学意义(P<0.05);针刺组大鼠浆皮质酮含量明显低于模型组,差异具有统计学意义(P<0.05);针刺组与空白对照组之间差异无统计学意义(P>0.05).模型组大鼠肾上腺皮质和髓质中的心房利钠肽水平均低于空白对照组,C型利钠肽水平均高于空白对照组,差异具有统计学意义(P<0.05);针刺组大鼠肾上腺皮髓质心房利钠肽明显高于模型组,C型利钠肽明显低于模型组,差异具有统计学意义(P<0.05);针刺组肾上腺髓质心房利钠肽、C型利钠肽与模型组之间差异无统计学意义(P>0.05).结论针刺可通过调节外周肾上腺髓质心房利钠肽、C型利钠肽水平,进而影响血浆皮质酮释放,抑制HPA轴活性,发挥抗焦虑作用.     

高寒草甸生态系统中轮牧实验的系统分析

采用高寒草甸放牧生态统的分室模型对高寒草甸生态系统轮牧实验进行系统分析。结果表明：１）轮牧并不一定能节约牧草。它可以在有限的草甸上控制绵羊生长速度或牧草消耗速度；使绵羊顺利地渡过冬春季，２）绵羊体重达到最大值的时间与放牧强度成反比。     

Identification and role of plasma membrane aquaporin in maize root

Using antiserum against expressed aquaporin fusion protein, GST-RD28, the distribution of aquaporin in the plasma membrane of maize root protoplasts has been examined under confocal laser scanning microscopy by indirect fluorescence staining. Results indicate that there are abundant aquaporins in maize roots, which are distributed in plasma membrane unevenly. Western blotting analysis of total protein solubilized from maize root plasma membrane shows that antiserum against GST-RD28 can cross-react with one protein around 55 Ku. Another 28 ku protein can also be detected when the concentration of SDS and DTT in SDS-PAGE sample buffer is increased. The 55 and 28 ku proteins may be dimeric and monomeric of aquaporin respectively. Functional experiments show that aquaporin blocker HgCl₂ and aquaporin antiserum can suppress the swelling of maize root protoplasts in hypotonic solution, indicating that aquaporin in plasma membrane of protoplast facilitates rapid transmembrane water flow.     

Experimental chimaeras--removal of reproductive barrier between sheep and goat

Following reciprocal embryo transfer between sheep and goats, the embryo of the foreign species is able to induce early pregnancy but the embryos do not survive beyond the first weeks of gestation. Similar results have been obtained from hybridization experiments on sheep and goats. While the causes for early embryonic death of hybrid eggs from donor sheep are unknown, the reciprocal event has been associated with immunological implications. As a means of overcoming the reproductive barrier between sheep and goats, we have transferred interspecific chimaeric embryos. We report here the birth of a goat kid from a sheep mother.     

Non-invasive prenatal measurement of the fetal genome

The vast majority of prenatal genetic testing requires invasive sampling. However, this poses a risk to the fetus, so one must make a decision that weighs the desire for genetic information against the risk of an adverse outcome due to hazards of the testing process. These issues are not required to be coupled, and it would be desirable to discover genetic information about the fetus without incurring a health risk. Here we demonstrate that it is possible to non-invasively sequence the entire prenatal genome. Our results show that molecular counting of parental haplotypes in maternal plasma by shotgun sequencing of maternal plasma DNA allows the inherited fetal genome to be deciphered non-invasively. We also applied the counting principle directly to each allele in the fetal exome by performing exome capture on maternal plasma DNA before shotgun sequencing. This approach enables non-invasive exome screening of clinically relevant and deleterious alleles that were paternally inherited or had arisen as de novo germline mutations, and complements the haplotype counting approach to provide a comprehensive view of the fetal genome. Non-invasive determination of the fetal genome may ultimately facilitate the diagnosis of all inherited and de novo genetic disease.