A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5’-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3’-UTR of 483bp and has more than 80% homology with that of other mammal oviductin...A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5’-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3’-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mous e cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn’t. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.展开更多
Rana kunyuensis is a species of brown frog that lives exclusively on Kunyu Mountain,Yantai,China.In the current study,a 279-bp cDNA sequence encoding a novel antimicrobial peptide (AMP),designated as amurin-9KY,was cl...Rana kunyuensis is a species of brown frog that lives exclusively on Kunyu Mountain,Yantai,China.In the current study,a 279-bp cDNA sequence encoding a novel antimicrobial peptide (AMP),designated as amurin-9KY,was cloned from synthesized double-strand skin cDNA of R.kunyuensis.The amurin-9KY precursor was composed of 62 amino acid (aa) residues,whereas the mature peptide was composed of 14 aa and contained two cysteines forming a C-terminal heptapeptide ring (Rana box domain) and an amidated C-terminus.These structural characters represent a novel amphibian AMP family.Although amurin-9KY exhibited high similarity to the already identified amurin-9AM from R.amurensis,little is known about the structures and activities of amurin-9 family AMPs so far.Therefore,amurin-9KY and its three derivatives (amurin-9KY1-3) were designed and synthesized.The structures and activities were examined to evaluate the influence of C-terminal amidation and the heptapeptide ring on the activities and structure of amurin-9KY..Results indicated that C-terminal amidation was essential for antimicrobial activity,whereas both C-terminal amidation and the heptapeptide ring played roles in the low hemolytic activity.Circular dichroism (CD) spectra showed that the four peptides adopted an α-helical conformation in THF/H2O (v/v 1∶1) solution,but a random coil in aqueous solution.Elimination of the C-terminal heptapeptide ring generated two free cysteine residues with unpaired thiol groups,which greatly increased the concentration-dependent anti-oxidant activity.Scanning electron microscopy (SEM) was also performed to determine the possible bactericidal mechanisms.展开更多
Gastrin is the main hormone responsible for the stimulation of gastric acid secretion;in addition,gastrin and its derivatives exert proliferative and antiapoptotic effects on several cell types.Gastrin synthesis and s...Gastrin is the main hormone responsible for the stimulation of gastric acid secretion;in addition,gastrin and its derivatives exert proliferative and antiapoptotic effects on several cell types.Gastrin synthesis and secretion are increased in certain situations,for example,when proton pump inhibitors are used.The impact of sustained hypergastrinemia is currently being investigated.In vitro experiments and animal models have shown that prolonged hypergastrinemia may be related with higher cancer rates;although,this relationship is less clear in human beings.Higher gastrin levels have been shown to cause hyperplasia of several cell types;yet,the risk for developing cancer seems to be the same in normo-and hypergastrinemic patients.Some tumors also produce their own gastrin,which can act in an autocrine manner promoting tumor growth.Certain cancers are extremely dependent on gastrin to proliferate.Initial research focused only on the effects of amidated gastrins,but there has been an interest in intermediates of gastrin in the last few decades.These intermediates aren't biologically inactive;in fact,they may exert greater effects on proliferation and apoptosis than the completely processed forms.In certain gastrin overproduction states,they are the most abundant gastrin peptides secreted.The purpose of this review is to examine the gastrin biosynthesis process and to summarize the results from different studies evaluating the production,levels,and effects of the main forms of gastrin in different overexpression states and their possible relationship with Barrett's and colorectal carcinogenesis.展开更多
Increasing amounts of antibiotic resistant bacteria have been an emergency problem. Antimicrobial peptides are promising antibiotic alternatives for broad-spectrum antimicrobial activity and nearly no drug resistance....Increasing amounts of antibiotic resistant bacteria have been an emergency problem. Antimicrobial peptides are promising antibiotic alternatives for broad-spectrum antimicrobial activity and nearly no drug resistance. Natto peptide was a new antimicrobial peptide which consisted of 45 amino acids. In this study, to improve the antimicrobial activity of Natto peptide, three repeats of encoding sequences were synthesized and cloned into a pET28 a(+) expression vector, and expressed in Escherichia coli as a soluble protein. Unexpectedly, the purified 3×Natto peptide exhibited antimicrobial activity against Listeria monocytogenes(50 μg/ml) and Salmonella enteriditis(30 μg/ml). Furthermore, the antibacterial spectrum of 3×Natto peptide was not affected by temperature, pH value and proteinase digestion. Taken together, this was the first study proving that 3×Natto peptide could be produced in E. coli as a kind of water dissolve protein, and has great potential for commercial application in the future.展开更多
This paper reported the identification of the primary structure of two peptides using nanoelectrospray tandem mass spectrometry(Nano-ESI MS/MS).Firstly,the relative molecular mass of two peptides were determined in ES...This paper reported the identification of the primary structure of two peptides using nanoelectrospray tandem mass spectrometry(Nano-ESI MS/MS).Firstly,the relative molecular mass of two peptides were determined in ESI-TOF MS mode.Then fragmentation ions were obtained by selecting [M+2H]2+ ion using tandem mass spectrometry(MS/MS).Finally,the primary structure of triptorelin is determined to be E’HWSYWLRPG’,of which the N-terminal is pyroglutamic acid (E’) and the C-termianl is glycinamide (G’).The primary structure of unknown peptide was identified to be T’VSP VWLPPSVY by sequence docking method,of which the N-terminal occurs threonine phosphorylation (T’) and the foruth proline is added with sodium ion (P).The data suggested that electrospray Tandem mass spectrometry technique have obvious advantage in analyzing the full sequence of modified or unknown peptides.展开更多
Directed peptides C-terminal modification enabled by the engineered biomolecular catalyst-peptide amidase 12 B has been achieved via computational protein engineering. The engineered enzyme exhibits great promising po...Directed peptides C-terminal modification enabled by the engineered biomolecular catalyst-peptide amidase 12 B has been achieved via computational protein engineering. The engineered enzyme exhibits great promising potential in the C-terminal modification of opioid peptides using prop-2-yn-1-amine(PYA) or prop-2-en-l-amine(PEA) as the nucleophile. A variety of opioid peptides could be readily functionalized at the C-terminal chain in high yield in a mild and selective manner. Notably, modified opioid peptides bearing alkynyl moiety could be further functionalized through well-established click reaction.展开更多
D-arginine oligomers have been widely used as intracellular delivery vectors both in in vitro andin vivo application.Nevertheless,their intemalization pathway is obscure and conficting resultshave been obtained concer...D-arginine oligomers have been widely used as intracellular delivery vectors both in in vitro andin vivo application.Nevertheless,their intemalization pathway is obscure and conficting resultshave been obtained concerning their intracellular distribution.In this study,we demonstrate that octa-D-arginine(r:)undergoes diffuse localization throughout the cytoplasm and nucleus even atlow concentrations and that rs(r:D-arginine)enters the cells via direct membrane translocation,unlike R:(R:L-arginine),of which endocytosis is the major internalization pathway.The observation that Rs and rg enter the cells through two clearly distinct internalization pathways suggests that the backbone stereochemistry affects the uptake mechanism of oligoarginines.展开更多
Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively...Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.展开更多
基金Supported by National Natural Science Foundation of China (39730460)National "973" Project (G1999055902)National Labora-
文摘A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5’-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3’-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mous e cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn’t. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.
基金supported by grants from the National Natural Science Foundation of China(31772455)Natural Science Foundation of Jiangsu Province(BK20160336and BK20171214)+1 种基金Natural Science Foundation of College in Jiangsu Province(16KJB350004)Suzhou Science and Technology Development Project(SYN201504 and SNG2017045)
文摘Rana kunyuensis is a species of brown frog that lives exclusively on Kunyu Mountain,Yantai,China.In the current study,a 279-bp cDNA sequence encoding a novel antimicrobial peptide (AMP),designated as amurin-9KY,was cloned from synthesized double-strand skin cDNA of R.kunyuensis.The amurin-9KY precursor was composed of 62 amino acid (aa) residues,whereas the mature peptide was composed of 14 aa and contained two cysteines forming a C-terminal heptapeptide ring (Rana box domain) and an amidated C-terminus.These structural characters represent a novel amphibian AMP family.Although amurin-9KY exhibited high similarity to the already identified amurin-9AM from R.amurensis,little is known about the structures and activities of amurin-9 family AMPs so far.Therefore,amurin-9KY and its three derivatives (amurin-9KY1-3) were designed and synthesized.The structures and activities were examined to evaluate the influence of C-terminal amidation and the heptapeptide ring on the activities and structure of amurin-9KY..Results indicated that C-terminal amidation was essential for antimicrobial activity,whereas both C-terminal amidation and the heptapeptide ring played roles in the low hemolytic activity.Circular dichroism (CD) spectra showed that the four peptides adopted an α-helical conformation in THF/H2O (v/v 1∶1) solution,but a random coil in aqueous solution.Elimination of the C-terminal heptapeptide ring generated two free cysteine residues with unpaired thiol groups,which greatly increased the concentration-dependent anti-oxidant activity.Scanning electron microscopy (SEM) was also performed to determine the possible bactericidal mechanisms.
基金Supported by Instituto de Salud Carlos Ⅲ with Grants FIS 08/1047 and CIBERehdInstituto de Salud Carlos Ⅲ FI10/00167, to Chueca E
文摘Gastrin is the main hormone responsible for the stimulation of gastric acid secretion;in addition,gastrin and its derivatives exert proliferative and antiapoptotic effects on several cell types.Gastrin synthesis and secretion are increased in certain situations,for example,when proton pump inhibitors are used.The impact of sustained hypergastrinemia is currently being investigated.In vitro experiments and animal models have shown that prolonged hypergastrinemia may be related with higher cancer rates;although,this relationship is less clear in human beings.Higher gastrin levels have been shown to cause hyperplasia of several cell types;yet,the risk for developing cancer seems to be the same in normo-and hypergastrinemic patients.Some tumors also produce their own gastrin,which can act in an autocrine manner promoting tumor growth.Certain cancers are extremely dependent on gastrin to proliferate.Initial research focused only on the effects of amidated gastrins,but there has been an interest in intermediates of gastrin in the last few decades.These intermediates aren't biologically inactive;in fact,they may exert greater effects on proliferation and apoptosis than the completely processed forms.In certain gastrin overproduction states,they are the most abundant gastrin peptides secreted.The purpose of this review is to examine the gastrin biosynthesis process and to summarize the results from different studies evaluating the production,levels,and effects of the main forms of gastrin in different overexpression states and their possible relationship with Barrett's and colorectal carcinogenesis.
基金Supported by Shandong Provincial Natural Science Foundation(ZR2018PC010)the Doctor Foundation of Binzhou University(2018Y09)Natural Science Program of Binzhou University(BZXYG1811)
文摘Increasing amounts of antibiotic resistant bacteria have been an emergency problem. Antimicrobial peptides are promising antibiotic alternatives for broad-spectrum antimicrobial activity and nearly no drug resistance. Natto peptide was a new antimicrobial peptide which consisted of 45 amino acids. In this study, to improve the antimicrobial activity of Natto peptide, three repeats of encoding sequences were synthesized and cloned into a pET28 a(+) expression vector, and expressed in Escherichia coli as a soluble protein. Unexpectedly, the purified 3×Natto peptide exhibited antimicrobial activity against Listeria monocytogenes(50 μg/ml) and Salmonella enteriditis(30 μg/ml). Furthermore, the antibacterial spectrum of 3×Natto peptide was not affected by temperature, pH value and proteinase digestion. Taken together, this was the first study proving that 3×Natto peptide could be produced in E. coli as a kind of water dissolve protein, and has great potential for commercial application in the future.
文摘This paper reported the identification of the primary structure of two peptides using nanoelectrospray tandem mass spectrometry(Nano-ESI MS/MS).Firstly,the relative molecular mass of two peptides were determined in ESI-TOF MS mode.Then fragmentation ions were obtained by selecting [M+2H]2+ ion using tandem mass spectrometry(MS/MS).Finally,the primary structure of triptorelin is determined to be E’HWSYWLRPG’,of which the N-terminal is pyroglutamic acid (E’) and the C-termianl is glycinamide (G’).The primary structure of unknown peptide was identified to be T’VSP VWLPPSVY by sequence docking method,of which the N-terminal occurs threonine phosphorylation (T’) and the foruth proline is added with sodium ion (P).The data suggested that electrospray Tandem mass spectrometry technique have obvious advantage in analyzing the full sequence of modified or unknown peptides.
基金supported by the National Natural Science Foundation of China(No.31601412)the 100 Talent Program grant and Biological Resources Service Network Initiative(No.ZSYS-012)grant from the Chinese Academy of Sciences(No.SKT1604)
文摘Directed peptides C-terminal modification enabled by the engineered biomolecular catalyst-peptide amidase 12 B has been achieved via computational protein engineering. The engineered enzyme exhibits great promising potential in the C-terminal modification of opioid peptides using prop-2-yn-1-amine(PYA) or prop-2-en-l-amine(PEA) as the nucleophile. A variety of opioid peptides could be readily functionalized at the C-terminal chain in high yield in a mild and selective manner. Notably, modified opioid peptides bearing alkynyl moiety could be further functionalized through well-established click reaction.
基金supported by the National High-Tech Research and Development Program of China(863 Program:2008AA02Z107)the National Natural Science Foundation of China(grant no.30800183)the Scientific Research Foundation for the Returned Overseas,Chinese Scholars,State Education Ministry(Y.-H.Zhang).
文摘D-arginine oligomers have been widely used as intracellular delivery vectors both in in vitro andin vivo application.Nevertheless,their intemalization pathway is obscure and conficting resultshave been obtained concerning their intracellular distribution.In this study,we demonstrate that octa-D-arginine(r:)undergoes diffuse localization throughout the cytoplasm and nucleus even atlow concentrations and that rs(r:D-arginine)enters the cells via direct membrane translocation,unlike R:(R:L-arginine),of which endocytosis is the major internalization pathway.The observation that Rs and rg enter the cells through two clearly distinct internalization pathways suggests that the backbone stereochemistry affects the uptake mechanism of oligoarginines.
文摘Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.
