The amino acid sequence of a double-headed trypsin inhibitor from the seeds of Momordica charantia Linn.Cucurbitaceae

A double-headed trypsin inhibitor(MCI-1)was isolated and purified from the seeds of Momordica charantia Linn.Cucurbitaceae,by using the trypsin-sepharose-4B affinity chroma- tography and CM-Sephadex-C50 ion exchange chromatography.It is composed of 77 amino acid residues:Asp_8 Thr_1 Ser_4 Glu_8 Pro_2 Gly_6 Ala_4 Cys_(14) Val_2 Met_4 Ile_8 Leu_1 Phe_1 His_3 Lys_ Arg_7. The amino acid sequence of MCI-1 was determined by sequencing the cyanogen bromide,tryptic and staphylococcus aureus V8 proteolytic peptides,then aligned by overlapped sequences.The result shows that MCI-1 contains 7 pairs of disulfide bonds,its sequence showed the high homology with those of “Bowman-Birk”inhibitors.About 50% trypsin inhibitory activity still remained after MCI-1 was cleavaged with cyanogen bromide.

Protective effects of tilapia(Oreochromis niloticus)skin gelatin hydrolysates on osteoporosis rats induced by retinoic acid

Tilapia skin gelatin hydrolysates(TSGH)were obtained by complex protease hydrolysis.The amino acid sequences of 50 peptides in TSGH were identified,and most of these peptides were found to contain the-Gly-Pro-sequence.The osteoporosis(OP)rat model induced by retinoic acid was prepared,and the effects of different doses of TSGH on OP in vivo were evaluated.Serum calcium(Ca)and phosphate(P),alkaline phosphatase activity,and osteocalcin levels in OP rats were regulated by TSGH.The bone length,dry weight index,maximum load,and Ca content of OP rats significantly increased by treatment TSGH in a dosedependent manner.Micro-CT images of the femurs and tibias of the rats indicated that the bone mineral density,cortical bone thickness,and cortical/trabecular bone area ratios were recovered and that OP symptoms were improved.Tartrate-resistant acid phosphate and hematoxylin-eosin staining showed that osteoclast numbers and histomorphological changes in the femurs in OP rats could be recovered by TSGH.

Analysis of the N Protein Sequence Variability in 13 Isolated PRRSV Strains from China

Object: To analyze porcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 infection cases via the N protein gene and its encoded amino acid sequence and to provide a theoretical basis for the epidemiological study, prevention and control of porcine reproductive and respiratory syndrome (PRRS). Methods: In clinically suspected PRRSV infections, viruses were isolated by extracting viral nucleic acid and amplifying the N protein gene by RT-PCR. Then, the product was purified and sequenced to acquire the whole gene sequence of the N protein and its encoded amino acid sequence. DNASTAR software was used to analyze the homology, the genetic evolution and the derivation of the variability of amino acids of the N protein gene from 13 PRRSV strains and classical domestic and foreign strains. Results: Among the thirteen strains of PRRSV isolated from this study, ten strains had the greatest homology with the JXA1 strain (98.9% - 100%), and they belonged to the sublineage 8.7. The remaining three strains had the greatest homology with the NADC30 strain (95.4% - 97.1%), and they belonged to lineage one. The analysis of the variability of N protein amino acids showed that there were high frequency mutations in the five loci of 13 isolated strains of PRRSV as follows: 15th amino acid (10/13), 46th amino acid (11/13), 91st amino acid (10/13), 109th amino acid (10/13), and 117th amino acid (10/13). Conclusion: In recent years, sublineage 8.7 was the dominant pedigree in field PRRSV epidemic strains in China with lineage one occupying a certain proportion of the field. Four high frequency mutations existed in N protein antigen epitopes of isolated strains from the region. The nuclear localization signal (NLS) structure, specifically the 46th amino acid residue of the N protein, was mutated and genetically stable.

Sequence Pattern Correlation of Amino Acid in Collision-induced Dissociation Electrospray Ionization Mass Spectrometry

A novel approach of sequence pattern correlation has been applied to predict an expected amino acid sequence from CID ESI MS spectra. The proposed approach deduces sequence patterns with no help from known protein database such that it is useful to identify an unknown peptide or new protein. The algorithm applies a cross correlation to match an experimental CID spectrum with predicted sequence pattern generated from fragmentation information. The fragmentation knowledge of both y series and other non y series are utilized to generate the predicted sequence patterns. In contrast to the normal de novo approach, the proposed approach is insensitive to mass tolerance and non susceptive to spectral integrality with no need for selection of a starting point.

A Novel Polypeptide from Cervus elaphus Linnaeus

A novel polypeptide having stimulant effect on some cell proliferation was isolated from the velvet antler (Cervus elaphus Linnaeus). The velvet antler polypeptide consists of a single chain of 32 amino acid residues. Amino acid sequence of the polypeptide was identified as: VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM.

Identification and Characterization of Peptide Mimics of Blood Group A Antigen

In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase （GST） fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.

A Novel Protein Found in Selenium-rich Silkworm Pupas

A novel protein was isolated and characterized in selenium-rich silkworm pupas. The peptide mass fingerprint of the protein was found to be new. Partial amino acid sequencing also confn-med to be a new protein. The novel protein had a molecular mass of about 80 kDa in the SDS-PAGE.

In silico Cloning and Bioinformatics Analysis of OsARAB-1 Gene from Rice

[ Objective] This study aimed to perform in silico cloning and bioinformatics analysis of OsARAB-1 gene from rice. [Method] Using NP 174188.1 as a query probe, the full-length cDNA sequence of OsARAB-1 gene was obtained by in silico cloning. The nucleotide sequence and protein sequence were analyzed by bioinformatics software. [Result] The full-length cDNA of OsARAB-1 gene was obtained. The coding sequence （CDS） of OsARAB-1 gene is 1 086 bp in length, encoding a protein of 361 amino acid residues. OsARAB-1 gene was located in the genome sequence NC 008398.2 （6 769 813 -6 773 213 bp segment） of rice chro- mosome 5 by in silico mapping. OsARAB-1 protein is an extracellular hydrophilic protein, which is relatively stable, slightly alkaline. The secondary structure of OsARAB-1 protein is mainly composed of or-helices and random coils. OsARAB-1 protein has two functional domains ： SGNH hydrolase-type esterase and GDSL li- pase. There are 21 phosphorylation sites and seven O-[3-GlcNAc glycosylation sites. The putative active-site amino acid residues are Ser34, Glyl07, Asn167, Asp333 and His336, respectively. OsARAB-1 protein has the closest genetic relationship with esterase subtype B4FM12 from maize. The expression of OsARAB-1 gene plays an important role in the development and morphogenesis of rice and is related with rice blast resistance. [ Conclusion] This study laid a solid foundation for cloning and functional identification of OsARAB-1 gene with experimental methods.

Genetic Characterization of Fusion Protein of Human Respiratory Syncytial Virus: Beijing

Objective Fusion protein is a subunit of the human respiratory syncytial virus(HRSV)and a potential vaccine candidate.Thus,a study on the genetic characteristics of F protein was considered important for further investigations in this field.The aim of this study was to determine the prevalence and genetic diversity of the F gene of HRSV infections in hospitalized pediatric patients in Beijing with acute lower respiratory tract infections and to compare the circulating genotypes that are currently found worldwide.Methods HRSV particles were amplified by RT-PCR and the PCR products were purified for sequencing.Further analysis was carried out by Bioedit and MEGA 3.0 biological software programs.Results Seventy-six samples(23.1%)were positive for HRSV.The percentage of cases in patients younger than 1year was 84.21%.Among the six Beijing isolates,four belonged to subgroup A,whose respective F genes shared97.0%-97.4%nucleotide sequence identity and 92.1%-93.0%amino acid sequence identity.The other two isolates belonged to subgroup B.Here,97.3%and 98.2%sequence identity were found at nucleotide and amino acid levels,respectively.Conclusions Phylogenetic analysis of nucleotide sequences revealed that those four isolates within subgroup A were monophyletic and closely related to each other,but those two within subgroup B distributed in two distinct clusters.Subgroup A and B strains co-circulated,indicating that two different transmission chains occurred in Beijing from 2003-2004.

Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea

Protease-producing bacteria and their extracellular proteases are key players in degrading organic nitrogen to drive marine nitrogen cycling and yet knowledge on both of them is still very limited. This study screened protease-producing bacteria from the South China Sea sediments and analyzed the diversity of their extracellular proteases at the family level through N-terminal amino acid sequencing. Results of the 16 S rRNA gene sequence analysis showed that all screened protease-producing bacteria belonged to the class Gammaproteobacteria and most of them were affiliated with different genera within the orders Alteromonadales and Vibrionales. The Nterminal amino acid sequence analysis for fourteen extracellular proteases from fourteen screened bacterial strains revealed that all these proteases belonged to the M4 family of metalloproteases or the S8 family of serine proteases. This study presents new details on taxa of marine sedimentary protease-producing bacteria and types of their extracellular proteases, which will help to comprehensively understand the process and mechanism of the microbial enzymatic degradation of marine sedimentary organic nitrogen.

Identification of Festuca arundinacea Schreb Cat1 Catalase Gene and Analysis of its Expression Under Abiotic Stresses

Ablotlc stresses, such as drought, high salinity, and cold/freezing, lead plants to produce excess reactive oxygen species. Catalase, a unique hydrogen peroxide-scavenging enzyme, plays a very Important role In plants. To characterize the catalase involved In plant response to ablotlc stresses, we constructed a cDNA library from 4℃-treated Festuca arundinacea Schreb seedlings and isolated a catalase gene from this library. The cDNA （FaCat1, 1 735 bp） contained an open reading frame of 1 479 bp. BLAST analysis Indicated that the deduced amino acid sequence showed 96% Identity with that from wheat TaCat1 and 87% Identity with that from maize ZmCat2. Northern blotting analysis showed an obvious Increase of FaCat1 transcripts In leaves In contrast with roots. Time-course analysis of the expression of FaCat1 in F. arundinacea leaves showed that FaCat1 expression was upregulated in cold- and salt-stressed leaves, with the FaCat1 transcripts accumulat-Ing mostly at 4 or 2 h after cold or salt stress, respectively. No significant changes in FaCat1 transcription were observed in dried leaves and inhibition of FaCat1 transcription was found In absclsic acid （ABA）-treated leaves, Indicating that the FaCat1 gene is differentially expressed during cold, high salt, drought, and ABA treatment In F. arundinacea leaves.

Characterization of hemagglutinin gene of influenza A virus subtype H9N2

OBJECTIVE: To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. METHODS: Viruses were passed in embryonated hen eggs, and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03) and Editseg (version 3.69) softwares. RESULTS: The amino acid sequences at the cleavage site between HA1 and HA2 domains of H9N2 viruses isolated in China are R-S-S-R. One pigeon strain contains seven potential glycosylation sites on the HA protein molecule, while all others have eight. There are 2 to 15 differences of amino acid sequences distributed at 24 different positions on the HA protein molecules among six H9N2 viruses. The H9N2 viruses with multiple lineages of HA genes were co-circulating in China recently. CONCLUSION: The highest possibility is that human influenza A (H9N2) virus was derived from Chicken H9N2 virus, and not derived from pigeon H9N2 virus. However, it is still unknown whether the H9N2 virus could transmit from person to person. The H9N2 viruses with multiple lineages of HA genes are co-circulating in China.

Identification in Lupin Seed of a Serine- Endopeptidase Activity Cleaving between Twin Arginine Pairs and Causing Limited Proteolysis of Seed Storage Proteins

The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the pres- ence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry anal- yses, These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-（2-AminoEthyl）Benzene-Sulphonyl Fluoride hy- drochloride （AEBSF）, leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans- EpoxysuccinyI-L-leucylamido（4-guanidino）butane （E64）, and ethylenediaminetetraacetic acid （EDTA）, thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Selection and determination of specific and protective antigens of Mycobacterium tuberculosis

Objective To select and identify the specific and protective antigens of Mycobacterium tuberculosis (M. tuberculosis) for searching a new approach to diagnose and treat tuberculosis Methods Extract and culture filtrates of M tuberculosis were obtained by ultrasonic treatment and millipore membrane filtration, respectively The protein samples were tested with monoclonal antibody (MAb) and patient sera The proteins showing positive reaction were sequenced on Beckman /LF 3200/peptide amino acid sequencer Results Proteins of M tuberculosis with molecular weight of 31?ku and 30?ku showed positive results when reacted with anti M tuberculosis MAb and sera of tuberculosis patients, but not with normal mouse serum and healthy human sera N terminal sequences of the 31?ku and 30?ku antigen were Ala Glu Val Asp Trp Leu Val Phe Ala Val and Phe Ser Arg Pro Gly Leu Pro Val Glu Try respectively Conclusion 31?ku and 30?ku proteins of M tuberculosis are immune protective proteins They play important or dominant roles in determination of immunorecognization

Purification,Characterization and ~1H NMR Resonance Assignment of an α-Like Neurotoxin BmK 16 from the Venom of Chinese Scorpion Buthus martensii Karsch

A natural scorpion toxin BmK 16 was purified for the first time from the venom of the Chinese scorpion Buthus martensii Karsch (BmK) by using combined gel filtration, ion exchange and reversed phase chromatography. The sequence of the N terminal 8 amino acid residues was determined by Edman degradation. Using the N terminal sequence as a tag, the database searching revealed a hit in the scorpion cDNA Bank. The sequence for N terminal 8 amino acid residues, molecular weight and amino acid compositions of BmK 16 were identical with the calculated values according to the first 64 residues' sequence of the precursor peptide alpha neurotoxin TX16 derived from the sequence of the cDNA AF156597 (EMBL). The sequence specific resonance assignment of BmK 16 was achieved and the intact sequence of BmK 16 was determined as followings: VRDAY IAKPH NCVYE CARNE YCNDL CTKNG AKSGY CQWVG KYGNG CWCKE LPDNV PIRVP GKCH. Furthermore, the results from the sequence homology analysis and the toxicity assays indicated that BmK 16 was an α like scorpion neurotoxin.

THE PRIMARY STRUCTURE OF ARROWHEAD PROTEINASE INHIBITOR

After the reduction and carboxymethylation of disulfide bonds, arrowhead proteinase inhibitors A and B were cleaved either by proteinases or by cyanogen bromide, the fractionated and purified peptides were then subjected to sequencing by a gas phase automatic sequencer, the primary structures were completed by the alignment of the peptides sequenced with overlapping peptides. Both inhibitors A and B consist of 150 amino acid residues with three pairs of disulfide bonds, share 90% homology in structure, and are markedly different from all other Ser proteinase inhibitors so far known. Hence, the arrowhead inhibitor may belong to a new inhibitor family. Based on their structure characteristics, it was deduced that both their two reactive sites might be located in the positions of Lys-Ser (45-46) and Arg-Tyr-Lys (77-79), respectively. Among 13 mutated residues in inhibitors A and B, the substitution of residue Arg in position 87 of inhibitor B for residue Leu in A might be the main cause of leading to

A novel peptide from Apis mellifera and solid-phase synthesis of its analogue

A novel peptide designated secapin-1, was purified and characterized from Apis mellifera. The molecular weight of 25 amino acid peptide secapin-1 was found to be 2821.5625 Da by ESI-FTICR-MS. It showed high identity to secapin. The sequence of secapin-1 was determined to be YIINVPPRCPPGSKFVKNKCRVIVP by automatic Edman degradation. A disulfide bond was formed between Cys9 and Cys20 residues. In addition, an analogue of secapin-1 was synthesized by solid phase peptide synthesis method. The synthesis product was successfully purified and identified to homogeneity by using a combination of SEC, IEC, and RP-HPLC techniques.