Structure of rat atrial natriuretic factor precursor deduced from cDNA sequence

The atrium of the heart contains peptides, termed atrial natriuretic factors ( ANFs ), diuretic and smooth-muscle-relaxing activities. In view of its potent effects on salt metabolism in the kidney and on vascular smooth muscle, ANF is considered to play an important part in the control of fluid volume and vascular function. Several different ANF peptides varying in size have been isolated and their amino acid sequences determined. Analysis of the sequences of the peptides suggests that they are derived by proteolysis from the same precursor. To examine this hypothesis, we have cloned cDNAs of the ANF precursor using rat atrial mRNA, determined its nucleotide sequence and deduced its amino acid sequence. The ANF precursor consists of 152 amino acid residues including a putative signal peptide sequence. This sequence contains the amino acid sequences of all the ANF peptides reported to date.     

mRNA sequence for human cardiodilatin-atrial natriuretic factor precursor and regulation of precursor mRNA in rat atria

The mammalian cardiac atrium has recently been shown to contain numerous peptides that exert marked effects on kidney function and vascular resistance. With one exception, the peptides have potent natriuretic and diuretic activities, and sequence similarities suggest that they may be derived from a common atrial natriuretic factor (ANF) precursor. The exception, cardiodilatin (CDD), differs from the ANF peptides in possessing potent vasorelaxant activity, but not natriuretic and diuretic activities. Here we report the cloning and sequence analysis of the cDNA for the human precursor protein (preproCDD-ANF) containing both the CDD and ANF sequences. The CDD sequence represents the N-terminal sequence preceded directly by a signal peptide, while the ANF sequence is present at the C-terminal end of the protein. Using hybridization analysis, we further show that the amount of rat preproCDD-ANF mRNA, which is synthesized selectively in the atria but not the ventricles, markedly decreases on water deprivation, suggesting that the water-electrolyte balance may be an important factor in the regulation of the expression of the preproCDD-ANF gene.     

Cloning and sequence analysis of cDNA encoding a precursor for human atrial natriuretic polypeptide

Recent identification of natriuretic-diuretic activity in peptides isolated from human and rat atrial tissue implicates them in the control of extracellular fluid volume and electrolytic homeostasis. The presence of multiple forms of the peptides ranging from 3,000 to 13,000 molecular weight (MW) suggests they may all derive from the same precursor. The established amino acid sequence of alpha-human atrial natriuretic polypeptide (alpha- hANP ), a 28-residue peptide with potent natriuretic activity, provided the means to elucidate the structure of the precursor for alpha- hANP and the gene encoding it. Here we report the cloning and sequence analysis of the cDNA of human atrial mRNA encoding a precursor of alpha- hANP . The cDNA encodes gamma-human atrial natriuretic polypeptide (gamma- hANP ) of 13,000 MW, whose C-terminal 28 amino acid residues may be processed as alpha- hANP .     

Atrial natriuretic factor--a circulating hormone stimulated by volume loading

The cardiocytes of mammalian cardiac atria contain granules very similar to those in endocrine cells. The number of these atrial granules is related directly to salt loading and blood volume. Furthermore, crude extracts of rat atria and granule preparations have powerful natriuretic and diuretic effects. These effects are mediated by peptides identified previously as atrial natriuretic factor (ANF). The peptides are derived from a common precursor, whose structure has been elucidated recently. Although there is indirect evidence from morphological studies that at least some of these peptides may be released into the blood and function as hormones, their presence in the blood has not yet been demonstrated. Here we describe a sensitive and specific radioimmunoassay for ANF and its stimulation on volume loading.     

Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth

The neurohumoral regulation of growth hormone secretion is mediated in part by two hypothalamic peptides that reach the anterior pituitary via the hypothalamo-hypophysial portal blood system. Somatostatin inhibits the release of growth hormone, whereas growth hormone-releasing factor (GRF) positively regulates both growth hormone synthesis and secretion. Two forms of human GRF, 40 and 44 amino acids long, have been characterized from extra-hypothalamic tumours as well as from the hypothalamus. Analysis of human GRF complementary DNA and genomic clones indicates that the GRF peptides are first synthesized as a 107- or 108-amino-acid precursor protein. To examine the physiological consequences of GRF expression, we have established strains of transgenic mice containing a fusion gene including the promoter/regulatory region of the mouse metallothionein-I (MT-I) gene and the coding region of the human GRF gene. We report that expression of the human GRF precursor protein in these animals results in measurable levels of human GRF and increased levels of mouse growth hormone in plasma and accelerated growth rates relative to control littermates. These results demonstrate a direct role for GRF in the positive regulation of somatic growth. Unexpectedly, female transgenic mice carrying the MT-GRF fusion gene are fertile, in contrast to female transgenic mice expressing human or rat growth hormone, which are generally infertile. These transgenic mouse strains should provide useful animal models for the study of several types of human growth disorders.     

Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases

Alpha atrial natriuretic peptide (alpha-ANP) and brain natriuretic peptide are homologous polypeptide hormones involved in the regulation of fluid and electrolyte homeostasis. These two natriuretic peptides apparently share common receptors and stimulate the intracellular production of cyclic GMP as a second messenger. Molecular cloning has defined two types of natriuretic peptide receptors: the ANP-C receptor of relative molecular mass (Mr) 60-70,000 (60-70 K), which is not coupled to cGMP production and may function in the clearance of ANP and the ANP-A receptor of Mr 120-140 K, which is a membrane form of guanylate cyclase in which ligand binding to the extracellular domain activates the cytoplasmic domain of the enzyme. Here we report the cloning and expression of a second human natriuretic peptide-receptor guanylate cyclase, the ANP-B receptor. The ANP-B receptor is preferentially activated by porcine brain natriuretic peptide rather than human alpha-ANP, whereas the ANP-A receptor responds similarly to both natriuretic peptides. These observations may have important implications for our understanding of the central and peripheral control of cardiovascular homeostasis.     

Inhibition of aldosterone production in the adrenal glomerulosa by atrial natriuretic factor

Several forms of the polypeptide atrial natriuretic factor (ANF) have been isolated recently from rat and human atria and identified; they are probably associated with the secretory granules of atrial tissue. The potent ability of ANFs to increase urine sodium content is mediated by their direct action on the kidney. We report here the high intrinsic activity of a synthetic replicate of one form of this molecule, ANF(8-33)(ref. 7), to inhibit directly basal aldosterone secretion and its ability to antagonize the stimulatory effects of adrenocorticotropin (ACTH) and angiotensin II (AN-II) on the secretion of aldosterone by rat adrenoglomerulosa cells in vitro. Our results suggest that ANF is of clinical importance in the management of aldosterone-dependent hypertension by modifying the adrenocortical response to endogenous ACTH and AN-II.     

Sequence of cDNA encoding human insulin-like growth factor I precursor

Somatomedins (SM) or insulin-like growth factors (IGF) constitute a heterogeneous group of peptides with important growth-promoting effects in vitro as well as in vivo. Amino acid sequences have been determined for only two of them, IGF-I and IGF-II, which are highly homologous. IGF-I, which is identical with SM-C, is composed of 70 amino acid residues and IGF-II contains 73 amino acids and may be identical with SM-A. Other peptides with different charge properties but with similar SM-like or insulin-like behaviour in biological and receptor assays, have been described but have not yet been fully characterized. The liver is known to be a major site of production of these peptides, but many other tissues--especially in the fetus--may synthesize them as well. We report here the nucleotide sequence of a human liver cDNA encoding the complete amino acid sequence of IGF-I. The IGF-I coding region is flanked by sequences encoding an amino-terminal peptide of at least 25 amino acid residues and a carboxyl-terminal peptide of 35 amino acids. This provides evidence that IGF-I is synthesized as a precursor protein and that formation of IGF-I from this precursor requires proteolytic processing at both ends.     

Cloning and sequence analysis of the cDNA for the rat atrial natriuretic factor precursor

Atrial extracts contain factors which induce potent natriuresis changes in renal haemodynamics, and relax pre-contracted vascular smooth muscle. Low-molecular-weight peptides which mimic these actions have now been purified by several groups, including ours (see accompanying paper), and higher-molecular-weight proteins with similar but less potent biological activities have also been identified and are presumed to be precursors. If released into the circulation, these peptides, collectively called atrial natriuretic factor (ANF), may play a significant part in blood-pressure homeostasis, regulation of extracellular fluid volume and as antagonists to the hypertensive effects of the renin-angiotensin system and other hormonal and neurotransmitter systems. We describe here the isolation and characterization of rat atrial cDNA clones which encode ANF. Nucleotide sequence analysis shows that auriculin corresponds to the 25 amino acids located close to the C-terminus of a 152-amino acid ANF precursor. Analysis of the in vitro translation products of precursor ANF mRNA suggests that multiple forms of the precursor may exist.     

The protein-coding sequence of the bovine ACTH-beta-LPH precursor gene is split near the signal peptide region

The pituitary hormones corticotropin (ACTH) and beta-lipotropin (beta-LPH) are formed from a large common precursor. Recently, we have elucidated the whole primary structure of the bovine ACTH-beta-LPH precursor (designated alternatively as preproopiocortin) by determining the nucleotide sequence of cloned DNA complementary to the mRNA coding for the precursor protein. The amino acid sequence assigned has disclosed a characteristic repetitive structure of the ACTH-beta-LPH precursor. The repetitive units of the precursor protein each contain a melanotropin (MSH) sequence (alpha-, beta- or gamma-MSH) as well as other peptide components such as beta-endorphin and corticotropin-like intermediate lobe peptide (CLIP). The repetitive units as well as their peptide components are each bounded by paired basic amino acid residues, which apparently represent the sites of proteolytic processing. Several studies have confirmed the translational initiation site and protein structure assigned (see also ref. 11 and refs therein). In view of the recent knowledge about the organization of eukaryotic genes (see refs 12, 13 for reviews), it would be of particular interest to investigate the relationship between the repetitive structure of the ACTH-beta-LPH precursor containing different functional components and the arrangement of the protein-coding sequence in its gene. We have now isolated and characterized bovine genomic DNA fragments encoding this precursor protein and have demonstrated that the protein sequence is encoded by two non-consecutive DNA segments. An intron (intervening sequence) of approximately 2.2 kilobase pairs separates the smaller exon (mRNA-coding sequence), which contains the gene sequence encoding the signal peptide, from the larger exon, which contains the gene sequence for most of the protein structure, including the known biologically active component peptides.     

A stop-codon mutation in the BRI gene associated with familial British dementia

Familial British dementia (FBD), previously designated familial cerebral amyloid angiopathy-British type, is an autosomal dominant disorder of undetermined origin characterized by progressive dementia, spasticity, and cerebellar ataxia, with onset at around the fifth decade of life. Cerebral amyloid angiopathy, non-neuritic and perivascular plaques and neurofibrillary tangles are the predominant pathological lesions. Here we report the identification of a unique 4K protein subunit named ABri from isolated amyloid fibrils. This highly insoluble peptide is a fragment of a putative type-II single-spanning transmembrane precursor that is encoded by a novel gene, BRI, located on chromosome 13. A single base substitution at the stop codon of this gene generates a longer open reading frame, resulting in a larger, 277-residue precursor. Release of the 34 carboxy-terminal amino acids from the mutated precursor generates the ABri amyloid subunit. The mutation creates a cutting site for the restriction enzyme XbaI, which is useful for detecting asymptomatic carriers. Antibodies against the amyloid or homologous synthetic peptides recognize both parenchymal and vascular lesions in FBD patients. A point mutation at the stop codon of BRI therefore results in the generation of the ABri peptide, which is deposited as amyloid fibrils causing neuronal disfunction and dementia.     

Structural identification of beta- and gamma-human atrial natriuretic polypeptides

Atrial natriuretic polypeptides (ANPs) of varying chain length have been identified recently in human and rat atrial tissue. Their potent natriuretic-diuretic activities indicate their key role in the regulation of extracellular fluid volume and electrolyte balance. Furthermore, human and rat cDNAs encoding their precursor have been cloned and identified. Natriuretic-diuretic activity in human atrial extract comprises three distinct components (alpha, relative molecular mass (Mr) approximately 3,000; beta, Mr approximately 6,000; gamma, Mr approximately 13,000). However, only the 3,000-Mr peptide, alpha-human atrial polypeptide (alpha-hANP), comprising 28 amino acids, has so far been identified. We report here the purification and sequence analysis of two novel hANPs of higher Mr, beta- and gamma-hANP, both of which exhibit natriuretic and hypotensive activity. gamma-hANP, composed of 126 amino acids, carries the alpha-hANP sequence at its carboxy terminus. The identification of gamma-hANP reveals that the peptide, being the largest form of hANP, is processed directly from a 151-residue precursor by removal of a 26-residue signal peptide. In contrast, beta-hANP (56 residues) comprises an anti-parallel dimer of alpha-hANP; such a dimeric peptide possessing bioactivity has never been found in the tissue as an endogenous entity.     

脑钠素的基因工程研究

脑钠素是利钠多肽家族成员之一 ,存在于人体的多种器官组织中 .脑钠素具有很强的利钠利尿、舒张血管和降低血压等生理功能 ,而脑钠素的改变与高血压、心脑血管病和肾病等许多疾病有关 .脑钠素具有潜在的药用开发价值和良好的应用前景 ,其开发通常采用基因工程手段 ,但至今尚未成功 .本文综合评述了脑钠素基因工程研究的历史与现状 ,全面分析了所存在的问题 ,并提出了相应的解决问题的方法 .     

Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity

Alzheimer's disease is characterized by cerebral deposits of amyloid beta-protein (AP) as senile plaque core and vascular amyloid, and a complementary DNA encoding a precursor of this protein (APP) has been cloned from human brain. From a cDNA library of a human glioblastoma cell line, we have isolated a cDNA identical to that previously reported, together with a new cDNA which contains a 225-nucleotide insert. The sequence of the 56 amino acids at the N-terminal of the protein deduced from this insert is highly homologous to the basic trypsin inhibitor family, and the lysate from COS-1 cells transfected with the longer APP cDNA showed an increased inhibition of trypsin activity. Partial sequencing of the genomic DNA encoding APP showed that the 225 nucleotides are located in two exons. At least three messenger RNA species, apparently transcribed from a single APP gene by alternative splicing, were found in human brain. We suggest that protease inhibition by the longer APP(s) could be related to aberrant APP catabolism.     

Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.     

Insulin-like growth factor II precursor gene organization in relation to insulin gene family

The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors I and II (IGF-I and IGF-II) and possibly the beta-subunit of 7S nerve growth factor, represents a group of structurally related polypeptides whose biological functions have diverged. The IGFs, or somatomedins, constitute a class of polypeptides that have a key role in pre-adolescent mammalian growth (see ref. 4 for review). IGF-I expression is regulated by growth hormone and mediates postnatal growth, while IGF-II appears to be induced by placental lactogen during prenatal development. The primary structures of both human IGFs have been determined and are closely related. A polypeptide highly homologous to human IGF-II is secreted by the rat liver cell line, BRL-3A. As this polypeptide, termed multiplication stimulating activity (MSA), differs from human IGF-II by only five amino acid residues, MSA probably represents the rat IGF-II protein. Using molecular cloning techniques, we have isolated cDNA and chromosomal genes coding for the MSA and human IGF-II precursors, respectively. Our data, presented here, indicate that both MSA and human IGF-II are synthesized initially as larger precursor molecules. The deduced preprohormones both have molecular weights (MWs) of 20,100 and contain C-terminal propeptides of 89 amino acid residues, which we have named E-peptides. The organization of the IGF-II precursor gene is discussed in relation to that of other insulin gene family members.     

Interleukin-2 production used to detect antigenic peptide recognition by T-helper lymphocytes from asymptomatic HIV-seropositive individuals

T lymphocytes from mice and healthy humans immunized against the human immunodeficiency virus (HIV) envelope have recently been shown to recognize two antigenic regions of the gp160 HIV-envelope protein which have been located on the basis of amphipathicity. In HIV-infected humans, T-cell proliferative responses are lost soon after infection. Here we demonstrate that interleukin-2 production is often retained even when proliferative activity is absent, and that it can be used to monitor T-helper cell responses by HIV-seropositive donors. We use this approach to investigate the T-helper cell response of 42 asymptomatic HIV-seropositive patients to four synthetic gp160 peptides and to influenza A virus, an antigen requiring intact CD4 T-helper cell function. As many as 67% of the HIV-seropositive donors who retain responsiveness to influenza A virus respond to a single peptide, and 85-90% responded to at least one of the peptides.     

酵母snoRNA基因簇启动子的比较分析

报道对酿酒酵母ｓｎｏＲＮＡ基因簇启动子序列的研究结果．通过计算机分析,发现酿酒酵母中Ｚ２Ｚ８和ＳｎＲ１９０Ｕ１４ｓｎｏＲＮＡ基因簇有相似的启动子结构．这两个ｓｎｏＲＮＡ基因簇都是由一个上游的启动子负责整个基因簇的转录,产生多个顺反子ｓｎｏＲＮＡ的前体,然后再加工成熟为各个独立的ｓｎｏＲＮＡ．在启动子保守序列ＴＡＴＡｂｏｘ的上游还发现２个ＲＡＰ１序列,表明ｓｎｏＲＮＡ基因簇的表达可以通过ＲＡＰ１元素与核糖体蛋白基因的表达协同调控．这是在ｓｎｏＲＮＡ基因的启动子中首次发现ＲＡＰ１调控元素．对ｓｎｏＲＮＡ基因簇的进化特点也进行了讨论．     

Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease

Several human bacterial pathogens, including the Gram-negative diplococcus Neisseria gonorrhoeae, produce extracellular proteases that are specific for human immunoglobulin IgA1. Immunoglobulin A (IgA) proteases have been studied extensively and the genes of some species cloned in Escherichia coli, but their role in pathogenesis remains unclear. Recently we derived a DNA fragment of 5 kilobases (kb) from N. gonorrhoeae MS11 directing extracellular active enzyme in E. coli. Although the mature enzyme of strain MS11 was shown to have a relative molecular mass of 106,000 (Mr 106K) in gels, the DNA sequence of this cloned fragment reveals a single gene coding for a 169K precursor of IgA protease. The precursor contains three functional domains, the amino-terminal leader which is assumed to initiate the inner membrane transport of the precursor, the protease, and a carboxyl-terminal 'helper' domain apparently required for extracellular secretion (excretion). Based on the structural features of the precursor, we propose a model in which the helper serves as a pore for excretion of the protease domain through the outer membrane. IgA protease acquires an active conformation as its extracellular transport proceeds and is released as a proform from the membrane-bound helper by autoproteolysis. The soluble proform further matures into the 106 K IgA protease and a small stable alpha-protein.