Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (In alpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparation for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaHdegrees and Delta(R,S)DeltaSdegrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect. (C) 2003 Elsevier B.V. All rights reserved.

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Identification and cloning of a trypsin inhibitor from skin secretions of Chinese red-belly toad Bombina maxima

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K-i value of 0.06 muM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K-i value of 1 muM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions. (C) 2002 Elsevier Science Inc. All rights reserved.

Molecular dynamics research of G249 and S249 substitutions of p53 protein.

,The molecular dynamics research of the core domain of p53 protein crystal structure shows that besides the stability in biochemistry this domain also shows a high stability in molecular mechanics. Based on that work, the residue R249 was substituted with amino acids Gly and Ser respectively, and molecular dynamics researches were performed separately. The results show that these substitutions cause a relax tendency between loop2 and 3 domains, leading to an alteration of the whole conformation of p53 core domain and ruining its stability. The results visually explains the mechanism of p53 changes in immunological and biochemical reactions, which are caused by 249 residue substitutions from 3-D structure variations.

An antimicrobial peptide with antimicrobial activity against Helicobacter pylori

An antimicrobial peptide named odorranain-HP was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLRASSVWGRKYYVDLAGCAKA. By BLAST search, odorranain-HP had si

Identification and molecular cloning of a novel neuromedin U analog from the skin secretions of toad Bombina maxima

Amphibian skin contains rich neuropeptides. In the present study, a novel neuromedin U (NmU) analog was isolated from skin secretions of Chinese red belly Load Bombina maxima. Being 17-amino acids long, its primary structure was established as DSSGIVGRPFFLFRPRN-NH2, in which the C-terminal 8-residue segment (FFLFRPRN) is the same as that of rat NmU, while the N-terminal part DSSGIVGRP shows a great sequence variation compared with those of NmU peptides from different resources. The peptide, named Bm-NmU-17, was found to elicit concentration-dependent contractile effects on smooth muscle of rat uterus horns. The cDNA Structure of the peptide, as obtained by a 3'-RACE strategy and subsequently cloning from a skin cDNA library, was found to contain a coding region of 438 nucleotides. The encoded precursor is composed of 145 amino acids with a single copy of Bm-NmU-17 located towards the C-terminus. The sequence of the peptide is preceded by a dibasic site (Lys-Arg) and followed by the sequence of Gly-Arg-Lys, providing the sites of cleavage and releasing of the mature peptide. (c) 2005 Elsevier B.V. All rights reserved.

A novel family of antimicrobial peptides from the skin of Amolops loloensis

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with beta-sheet in water, Tris-HCl or Tris-HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature amolopins are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. (C) 2008 Elsevier Masson SAS. All rights reserved.

A novel antimicrobial peptide from amphibian skin secretions of Odorrana grahami

A novel antimicrobial peptide named odorranain-NR was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLSGILGAGKHIVCGLTGCAKA. Odorranain-NR was classified into a novel family of antimicrobial peptide although it shared similarity with amphibian antimicrobial peptide family of nigrocin. Odorranain-NR has an unusual intramolecular disulfide-bridged hexapeptide segment that is different from the intramolecular disulfide-bridged heptapeptide segment at the C-terminal end of nigrocins. Furthermore, the -AKA fragment at the C-terminal of odorranain-NR is also different from nigrocins. Three different cDNAs encoding two odorranain-NR precursors and only one mature odorranain-NR was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms except Escherichia coli (ATCC25922). Its antimicrobial mechanisms were investigated by transmission electron microcopy. odorranain-NR exerted its antimicrobial functions by various means depending on different microorganisms. (C) 2008 Elsevier Inc. All rights reserved.

Two serine protease inhibitors from the skin secretions of the toad, Bombina microdeladigitora

Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, Bombina microdeladigitora. The cDNAs encoding BMSIs were cloned from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI1 and BMSI2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 mu M and 0.15 mu M, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures. (c) 2007 Elsevier Inc. All rights reserved.

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15,536 Da) and beta (14,873 Da). The mature amino acid sequences of the a (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers Of (alphabeta)(2) and (alphabeta)(4) with molecular weights of 63,680 and 128,518 Da, respectively. The (alphabeta)(2) complex is stabilized by an interchain disulfide bridge between the two alphabeta-heterodimers, whereas the stabilization of the (alphabeta)(4) complex seems to involve non-covalent interactions between the (alphabeta)(2) complexes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Evolution of structures and specific recognitions in aminoacyl-tRNA synthetases

The aminoacyl-tRNA synthetases (AARS) are very important during the protein biosynthesis, which can make the gene sequence be accurately translated into the protein sequence by the specific recognition between AARS and tRNA/amino acids. However, the recog

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCSC2, TCSC4, and TCSC14) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCSC19aa and TCSKDEL having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS. (C) 2003 Elsevier Science (USA). All rights reserved.

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG(20)k-S7C, PEG(20)k-K173C, and PEG(20)k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life. (C) 2004 Elsevier Inc. All rights reserved.

适合脑内多种神经活性物质免疫组化研究的复合固定

　寻找适合脑内多种神经活性物质免疫组织化学研究的固定方案。方法　以胆碱乙酰化酶、P 物质和γ- 氨基丁酸为递质合成酶、多肽及氨基酸递质的代表,以大鼠隔O斜带O基底核此三类阳性神经元的数量、染色和形态 变化为依据,检测了一系列复合固定配方对此三类抗原免疫组化的影响。结果　经0. 2 %戊二醛、4 %多聚甲醛和 0. 2 %苦味酸灌注固定和4 %多聚甲醛后固定,并经1 %硼氢化钠处理的脑组织,能很好地显示该三类神经元。此结 果还得到猫脑免疫电镜研究的支持。结论　有可能设计一种满足脑内尽可能多种类递质及合成酶免疫组化光、电 镜研究所需的复合固定配方,以期在同一实验动物获得最大程度的化学神经解剖学信息。