New O-superfamily conotoxins fromConus striatus inhabited near Chinese Hainan Island

Conotoxins are short peptide-toxins with specific targets and large diversity. They are useful in analgesia, neuroprotection, detection of some kinds of deseases, and receptor and ion channel study. In order to explore the conotoxin resourses of Chinese oceans, rapid amplification of 3′ cDNA ends (RACE) method was utilized to systemically analyze the O-superfamily conotoxin content ofConus striatus inhabited near Chinese Hainan Island. Six new O-superfamily conopeptides were identified, one of which is highly homologous to MVIIA, an N-type calcium channel antagonist.     

Immunoglobulin-like nature of the alpha-chain of a human T-cell antigen/MHC receptor

Although the receptor with which T cells bind specific antigen can, like immunoglobulin, distinguish between antigens which differ only slightly in structure, it is unique in recognizing antigen only in conjunction with one of the self proteins of the major histocompatibility complex (MHC restriction). The receptor was identified and characterized in mouse and man by using monoclonal antibodies to receptor idiotypes, and consists of two disulphide-linked polypeptides, and acidic alpha-chain and a neutral to slightly basic beta-chain. Peptide maps have shown that, like immunoglobulin, both chains vary for receptors of different specificities. T-cell-derived cDNA clones have recently been identified in mouse and man encoding immunoglobulin-like molecules. These were identified as derived from beta-chain genes through a partial N-terminal protein sequence of the beta-chain isolated from a human T-cell tumour. We have now purified the alpha- and beta-chains of the receptor of the human T-cell leukaemia line HPB-MLT, and have determined the amino acid sequence of several tryptic peptides derived from each chain. Our results further confirm that the previously reported cDNA clones encode beta-chains. The sequence of the alpha-chain peptides identify this as another immunoglobulin-like polypeptide chain. Particularly striking was an alpha-chain peptide with high homology to the conserved portion of the immunoglobulin J segment and T-cell receptor beta-chains. Surprisingly, the alpha-chain peptides show little similarity to the sequence predicted by two overlapping putative murine alpha-chain cDNA clones.     

Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter.

In mammalian cells, short peptides derived from intracellular proteins are displayed on the cell membrane associated with class I molecules of the major histocompatibility complex (MHC). The surface presentation of class I-peptide complexes presumably alerts the immune system to intracellular viral protein synthesis. Peptides derived from the cytosol must reach the cisternae of the endoplasmic reticulum where they are required for the assembly of stable class I molecules, and it has been proposed that the products of the two MHC-encoded ATP-binding cassette (ABC) transporter genes function to deliver the peptides across the membrane of the endoplasmic reticulum. This idea is supported by experiments in which transfection of a human cell line defective in class I expression with a complementary DNA of one of these genes restored cell surface expression levels. Here we show that the complete phenotype of the mouse mutant cell line RMA-S, in which lack of surface expression of stable class I molecules correlates with an inability to present viral peptides originating in the cytosol, is repaired by the cDNA of the other transporter gene. These results are consistent with the possibility that the two transporter polypeptides form a heterodimer.     

Mimicking the alloantigenicity of proteins with chemically synthesized peptides differing in single amino acids

Recent studies have shown that short chemically synthesized peptides very often induce antibodies which react with the cognate sequence in the intact folded protein. Since such antibodies react with known regions of proteins, they are of predetermined specificity and offer a precision not previously possible with immunological probes. A basic concept emerging from the use of such antibodies in viral systems is that the differential immunogenicity of closely related proteins can be mimicked by short peptides which span the regions of sequence variation. To generalize this concept, we have studied the two Thy-1 proteins which vary by only a single amino acid. Chemically synthesized peptides differing in only one out of 19 amino acids were able to induce allospecific antisera. Thus, single amino acid changes have similar effects on the immunogenicity of proteins and small peptides, even though the latter are free from constraints provided by neighbouring structures in the tertiary configuration of the intact folded proteins.     

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.     

用响应面法优化酶水解法制备葵花盘小分子肽

为提取并利用葵花盘中具有降尿酸和溶解痛风石作用的小分子肽, 以小分子肽得率为指标, 通过酶水解法优化提取条件, 确定碱性蛋白酶为最理想的酶. 通过单因素实验考察料液比、 时间、 温度、 pH值和加酶量对小分子肽得率的影响, 利用响应面Box-Behnken实验, 优化酶水解法提取葵花盘小分子肽的工艺, 得到其最佳工艺条件为： 加入碱性蛋白酶, m(料)∶V(液)=1∶15, 酶的用量为2%, pH=9.0, 温度为55 ℃, 时间为2 h, 葵花盘小分子肽的得率最高为(42.41±0.01)%.     

Strategy for rapid immobilization of prey by a fish-hunting marine snail

Some venomous animals capture prey with remarkable efficiency and speed. The purple cone, Conus purpurascens, uses two parallel physiological mechanisms requiring multiple neurotoxins to immobilize fish rapidly: neuromuscular block, and excitotoxic shock. The latter requires the newly characterized peptide kappa-conotoxin PVIIA, which inhibits the Shaker potassium channel 2-4, and beta-conotoxin PVIA5, which delays sodium-channel inactivation. Despite the extreme biochemical diversity in venoms, the number of effective strategic alternatives for prey capture are limited. How securely prey is initially tethered may strongly influence the venom strategy evolved by a predator.     

Cloning and expression of a cDNA encoding an endothelin receptor

Endothelins are a newly described peptide family consisting of three peptides (ET-1, ET-2 and ET-3) which are the most potent vasoconstrictive peptides known. They are crucial in the regulation of vascular smooth muscle tone. The diverse functions of endothelins are thought to be mediated by interaction with many different receptors coupled to the inositol phosphate/calcium ion messenger pathway. However, because of the structural resemblance of the three peptides, the presence and nature of multiple endothelin receptors remain to be elucidated. We report here the cloning of a complementary DNA encoding a bovine endothelin receptor, which has a transmembrane topology similar to that of other G protein-coupled receptors and shows specific binding, with the highest selectivity to ET-1 in animal cells transfected with the cloned cDNA. This receptor messenger RNA is widely distributed in the central nervous system and peripheral tissues, particularly in the heart and lung. Our results support the view that there are other receptor subtypes.     

高效液相色谱法分离大豆蛋白肽

本文采用Alcalase碱性内切蛋白酶为工具酶水解大豆蛋白,通过实验选择最佳水解条件。采用超滤以及凝胶过滤色谱法(Sephadex G 15)分离出分子量1000以下的小肽并收集,将收集到的不同功能性目标肽段在最佳色谱条件下进行分离。     

用响应面法优化酶水解法制备葵花盘小分子肽

为提取并利用葵花盘中具有降尿酸和溶解痛风石作用的小分子肽, 以小分子肽得率为指标, 通过酶水解法优化提取条件, 确定碱性蛋白酶为最理想的酶. 通过单因素实验考察料液比、 时间、 温度、 pH值和加酶量对小分子肽得率的影响, 利用响应面Box-Behnken实验, 优化酶水解法提取葵花盘小分子肽的工艺, 得到其最佳工艺条件为： 加入碱性蛋白酶, m(料)∶V(液)=1∶15, 酶的用量为2%, pH=9.0, 温度为55 ℃, 时间为2 h, 葵花盘小分子肽的得率最高为(42.41±0.01)%.     

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 .     

A linguistic model for the rational design of antimicrobial peptides

Antimicrobial peptides (AmPs) are small proteins that are used by the innate immune system to combat bacterial infection in multicellular eukaryotes. There is mounting evidence that these peptides are less susceptible to bacterial resistance than traditional antibiotics and could form the basis for a new class of therapeutic agents. Here we report the rational design of new AmPs that show limited homology to naturally occurring proteins but have strong bacteriostatic activity against several species of bacteria, including Staphylococcus aureus and Bacillus anthracis. These peptides were designed using a linguistic model of natural AmPs: we treated the amino-acid sequences of natural AmPs as a formal language and built a set of regular grammars to describe this language. We used this set of grammars to create new, unnatural AmP sequences. Our peptides conform to the formal syntax of natural antimicrobial peptides but populate a previously unexplored region of protein sequence space.     

多肽的表面展示与结构库

表面展示是一种新的基因操作技术,它使表达的多肽以融合蛋白形式展现在噬菌体或细胞表面,保持相对独立的空间结构和生物活性。该技术可用于研究多肽(蛋白质)的性质、相互识别和作用,并据此从巨大展示库中选择特定靶功能的多肽结构。常用丝状噬菌体、T₄噬菌体、λ噬菌体以及细胞构建表面展示系统。表面展示库包括重组噬菌体抗体库、随机短肽库、多肽构象库、cDNA展示库和基因突变体展示库。表面展示技术可用于人工抗体和疫苗的制备、抗原决定簇的定位、蛋白质相互作用位点的确定、特异调节分子的分离、细胞表面工程的研究、多肽药物的研制,以及生物分子实验定向进化等研究。     

尼罗罗非鱼Hepcidin基因结构与序列分析

Hepcidins是一类具有调节铁代谢功能的抗菌肽.利用RT-PCR、RACE和LA等技术,以尼罗罗非鱼[Oreochro-mis niloticus(Linnaens)]肝脏中分离和克隆到Hepcidin基因.其全长cDNA为505 bp(不包括polyA),5′端非翻译区有85 bp,3′非编码区为156 bp,阅读框为264 bp.其编码的氨基酸包括信号肽和前体肽等,前体肽进一步酶解产生22个氨基酸的活性肽,该活性肽具有Hepcidin基因家族特有8个半胱氨酸的保守序列.罗非鱼的Hepcidin基因结构包含3个外显子和2个内含子.本研究为今后阐明Hepcidin基因表达特性、表达产物理化性质、抗菌活性及其相关功能等奠定基础.     

Identification and functional analysis of 23 novel box C／D snoRNAs from Oryza sativa

In a cDNA library generated from rice small nuclear RNAs,30box C/D small nucleolar RNAs (snoRNAs) were identiffied through preliminary screen.Except 7 known snoRNAs such as U14,all snoRNAs were identified in rice for the first time experimentally.Among the 23 novel snoRNAs,11 snoRNAs appear rice-specific,6 snoRNAs are unique to plants,the remaining 6 snoRNAs have their counterparts in both Arabidopsis and yeast or mammals according to the conserved antisense sequencs that guide 2‘-O-ribose methylation of rRNA,17 of the 23 novel snoRNAs were predicted to guide 24 2‘‘-O-ribose methylations at the specificsites of rice 5.8S,18S,25S rRNAs,among which 19 methylated sites were determined by primer extension at low dNTP concentrations.The remaining 6 snoRNAs devoid of rRNA antisense elements may represent novel snoRNA species in rice.The results show that constructing a cDNA library from small nuclear RNAs is an effective experimental approach for novel snoRNA is identification.The novel snoRNAs are important in elucidating the genomic organization and expression of plant snoRNA genes and the mechanism through which 2‘‘-O-ribose methylations took place in rRNAs.     

Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides

Naturally occurring peptides with biological actions have in most cases been detected by observing their biological activities in crude extracts and their isolation has been followed using bioassays. As a complement to the classical biological detection systems, we have proposed a chemical detection system based on fragmentation of peptides in tissue extracts followed by identification of certain of these peptide fragments having distinct chemical features. One such chemical feature is the C-terminal amide structure which is characteristic of many biologically active peptides. We have devised a chemical assay method for peptides having such a structure and have found several previously unknown peptide amides in procine upper small intestinal tissues. We report here the isolation and characterization of two of them, designated PHI and PYY. PHI is related to secretin, vasoactive intestinal polypeptide (VIP, glucagon and gastric inhibitory polypeptide (GIP); PYY is related to the pancreatic polypeptide and to neurotensin. Both peptides exhibit biological activities and appear to be present not only in the intestine but also in brain.     

Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor.

The nucleotide sequence of a 1,091-base pair cloned cDNA insert encoding bovine corticotropin-beta-lipotropin precursor mRNA is reported. The corresponding amino acid sequence indicates that the precursor protein consists of repetitive units and includes a third melanotropin sequence in its cryptic portion. Pairs of lysine and arginine residues separate the component peptides of the precursor.     

Effect of New O-Superfamily Conotoxin on Voltage-Activated Currents of Hippocampal Neurons

The effects of a new O-superfamily conotoxin, SO3, on sodium current (INa), transient A-type potassium currents (IA), and delayed rectified potassium currents (IK), were examined in cultured rat hippocampal neurons using the whole-cell patch clamp technique. Addition of SO3 caused a concentration-dependent, rapidly developing, and reversible inhibition of voltage-activated currents. The IC50 values for the blockage of INa, IA, and IK were calculated as 0.49, 33.9, and 7.6 μmol/L, respectively. The determined Hill coefficients were 1.7, 0.6, and 1.2, respectively. These results indicate that SO3 can selectively inhibit neuronal sodium and potassium currents.     

甘蓝磷酯酶D HKD2功能区基因片段的克隆与反义表达载体的构建

以甘蓝(Brassiaca oleracea)为材料，取幼叶分离mRNA，反转录合成cDNA，以cDNA第一链为模板，通过PCR扩增，获得甘蓝磷酯酶D(Phosphorate　Lipid　Dehydrolase，PLD)HKD2功能区的基因片段．对其进行Blast分析，结果表明，分离的目的片段核苷酸序列与Genbank中报道的甘蓝PLD基因相比同源率为99．7％，只有2个碱基发生改变．将得到的PLD基因片段插入植物表达载体pAT940，构建了PLD基因反义表达载体pATC—rPLD，为下一步进行抗逆转基因作物选育打下基础．     

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.