Biomedical subjects

Song-Ping Liang

Publications and source records attributed to Song-Ping Liang.

At least 19 recordsLinked to original sources

PubMed2006

Nuclear magnetic resonance studies on huwentoxin-XI from the Chinese bird spider Ornithoctonus huwena: 15N labeling and sequence-specific 1H, 15N nuclear magnetic resonance assignments.

Huwentoxin-XI purified from the Chinese bird spider Ornithoctonus huwena is a toxin with both antiprotease activity and potassium channel blocking activity. To determine its solution structure, huwentoxin-XI was expressed in a yeast eukaryotic expression system and studied by NMR. The 15N labeling strategy was used to facilitate the process of resonance assignments. The nearly complete sequence-specific assignments of proton and nitrogen resonances were obtained by analyzing a series of two-dimensional (2D) and three-dimensional (3D) spectra, including DQF-COSY, TOCSY, NOESY, 15N-1H HSQC, 15N-1H HNHA, 15N-1H HNHB, 15N-1H TOCSY-HSQC and 15N-1H NOESY-HSQC spectra. Secondary structure analysis of huwentoxin-XI showed that it mainly contains an N-terminal 310-helix from Thr3 to Arg5 and a C-terminal alpha-helix from Gln45 to Cys52, plus a triple-stranded antiparallel beta-sheet of Glu18-Asn23, Thr26-Ile31 and Asn40-Lys41. These studies provide a solid basis for the final structure determination of huwentoxin-XI.

Animals↗

PubMed2005

DEPD: a novel database for differentially expressed proteins.

SUMMARY: The Differentially Expressed Protein Database was designed to store the output of comparative proteomics studies and provides a publicly available query and analysis platform for data mining. The database contains information about more than 3000 differentially expressed proteins (DEPs) manually extracted from the published literature, including relevant biological, experimental and methodological elements. Tools for visualization and functional analysis of DEPs are provided via a user-friendly webinterface. AVAILABILITY: http://protchem.hunnu.edu.cn/depd/.

Computational Biology↗

PubMed2005

Antinociceptive effects of intrathecally administered huwentoxin-I, a selective N-type calcium channel blocker, in the formalin test in conscious rats.

The present study was undertaken to elucidate the antinociceptive effect of intrathecal administration of huwentoxin-I (HWTX-I), a N-type calcium channel blocker from the venom of the Chinese bird spider Ornithoctonus huwena (Wang) [=Selenocosmia huwena wang], by comparison with omega-Conotoxin-MVIIA (omega-CTX-MVIIA) and morphine hydrochloride in the formalin test in conscious rats. Similar to omega-CTX-MVIIA and morphine, intrathecal pre-treatment with HWTX-I resulted in suppression of nociceptive behavior in a dose-dependent manner. The ED50 values of HWTX-I and omega-CTX-MVIIA were 0.28 and 0.19 microg/kg, respectively. It was also found that, at lower doses (0.1 and 0.5 microg/kg), the antinociceptive effect of HWTX-I was identical to that of omega-CTX-MVIIA, while omega-CTX-MVIIA acted more remarkably than HWTX-I at higher dose (1.0 microg/kg). However, the antinociception induced by omega-CTX-MVIIA were companied with motor dysfunction, and these side-effects became more evident with the doses of omega-CTX-MVIIA increasing. In contrast, HWTX-I did not show these side-effects at the doses of 0.5-1.0 microg/kg. Compared with HWTX-I and omega-CTX-MVIIA, the analgesic effect of intrathecal morphine hydrochloride was initiated faster, but lasted for a shorter time (about 2-3 h at 1.0 microg/kg) than that of HWTX-I and omega-CTX-MVIIA (about 4- 5 h at 1.0 microg/kg). Therefore, the present results show that, like omega-CTX-MVIIA, the intrathecal administration of HWTX-I is effective in antinociception in the rat model of the formalin test.

Analgesics↗

PubMed2005

Sequence-specific assignment of 1H-NMR resonance and determination of the secondary structure of Jingzhaotoxin-I.

Jingzhaotoxin-I (JZTX-I) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by d alphaN, d alphadelta, d betaN and d NN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel beta-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I (HWTX-I) and hainantoxin-IV (HNTX-IV), whose structures in solution have previously been reported.

Amino Acid Sequence↗

PubMed2005

[Solid-phase synthesis and biological characterization of S12A-HNTX-IV and R29A-HNTX-IV: two mutants of hainantoxin-IV].

Hainantoxin-IV (HNTX-IV) purified from the venom of the spider Selenocosmia hainana is a potent antagonist that acts on tetrodotoxin-sensitive (TrX-S) sodium channels. It is a 35-residue polypeptide and includes three disulfide bridges. In order to investigate the structure-function relationship of HNTX-IV, two mutants (S12A-HNTX-IV and R29A-HNTX-IV) of HNTX-TV in which Ser12 and Arg29 were replaced by Ala respectively, were synthesized by solid-phase Fmoc chemistry, followed by oxidative refolding of purified peptides under the optimal conditions. The synthetic mutants were analyzed by MALDI-TOF mass spectrometry, nuclear magnetic resonance spectroscopy (NMR) and electrophysiological experiments for molecular weight, conformation and physiological activity, respectively. The results show that the mutants and native HNTX-IV (nHNTX-IV) have almost identical three-dimensional structures. The bioactivity level of S12A-HNTX-IV is also about the same as that of nHNTX-IV, suggesting that Ser12 does not play any important role for the bioactivity of this toxin. The bioactivity of R29A-HNTX-IV is reduced by at last 155 times, indicating that Arg29 is a key residue relative to the bioactivity of HNTX-IV. It is presumed that the decrease in activity of R29A-HNTX-IV is due to the changes of the property in the binding site rather than the change in the basic conformation of the molecule.

Amino Acid Substitution↗

PubMed2004

De novo sequencing of tryptic peptides sulfonated by 4-sulfophenyl isothiocyanate for unambiguous protein identification using post-source decay matrix-assisted laser desorption/ionization mass spectrometry.

A simple method of solid-phase derivatization and sequencing of tryptic peptides has been developed for rapid and unambiguous identification of spots on two-dimensional gels using post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The proteolytic digests of proteins are chemically modified by 4-sulfophenyl isothiocyanate. The derivatization reaction introduces a negative sulfonic acid group at the N-terminus of a peptide, which can increase the efficiency of PSD fragmentation and enable the selective detection of only a single series of fragment ions (y-ions). This chemically assisted method avoids the limitation of high background normally observed in MALDI-PSD spectra, and makes the spectra easier to interpret and facilitates de novo sequencing of internal fragment. The modification reaction is conducted in C(18) microZipTips to decrease the background and to enhance the signal/noise. Derivatization procedures were optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. The MALDI-PSD mass spectra of two model peptides and their sulfonated derivatives are compared. For some proteins unambiguous identification could be achieved by MALDI-PSD sequencing of derivatized peptides obtained from in-gel digests of phosphorylase B and proteins of hepatic satellite cells (HSC).

Amino Acid Sequence↗

PubMed2004

[Differential analysis of two-dimension gel electrophoresis profiles of human lung squamous carcinoma and tumor-adjacent tissue].

BACKGROUND & OBJECTIVE: Carcinogenesis of lung squamous carcinoma is a complex process involving multiple events and steps. Though some molecular pathogenesis studies on human lung cancer have been undertaken successfully in gene (DNA) and transcription (mRNA) levels, the carcinogenic mechanism is still unclear. At present, there is no special molecular marker for early-stage diagnosis and prognosis evaluation. The objective of this study was to establish two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue, and to identify differential expression proteins. METHODS: The total proteins of human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue were separated by immobilized pH gradient (IPG)-based two-dimensional gel electrophoresis (2-DE). The differential expression proteins were analyzed using image analysis software, then identified using mass spectrometry and database searching. RESULTS: (1) For tumor tissues, the average protein spots of 3 gels were 1349+/-67; and 1235+/-48 spots were matched with the average matching rate of 91.5%. For control, the average protein spots of 3 gels were 1297+/-73; and 1183+/-56 spots were matched with the average matching rate of 91.2%. The average position deviation of matched spots was 0.873+/-0.125 mm in IEF direction, and 1.025+/-0.213 mm in SDS-PAGE direction. (2) A total of 1069+/-45 spots were matched between the electrophoretic maps of 15 human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue. Forty differential proteins were identified by peptide mass fingerprint(PMF), some proteins were the products of oncogenes, and the others involve in the regulation of cell cycle and signal transduction. CONCLUSION: In this study, the well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were established and certain differential proteins were characterized. These data will be helpful for screening the biomarker to further study on human lung squamous carcinoma.

Amino Acid Sequence↗

PubMed2004

[Establishment of protein profile of human small cell lung cancer cell line NCI-H446].

BACKGROUND & OBJECTIVE: Small cell lung cancer (SCLC) is particularly aggressive, and characterized by rapid growth and early metastasis. At present, there is no data concerning SCLC two-dimensional polyacrylamide gel electrophoresis (2-DE) reference map,and its protein profiles in public databases. This study was to establish a well-resolved, reproducible 2-DE map of proteome in SCLC cell line NCI-H446, and analyze its protein profiles. METHODS: Two-DE was applied to separate the total proteins of NCI-H446 cells, which were then silver-stained in the gel. Well-separated protein spots were selected from the gel by ImageMaster 2D analysis system. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), peptide map fingerprinting (PMF),and database searching were used to identify the protein spots. RESULTS: Clear,well-resolved, reproducible 2-DE patterns of proteome in NCI-H446 cells were obtained. The average protein spots of 3 gels were 1506+/-74; and 1412+/-56 spots were matched with an average matching rate of 93.4%. The average deviation of spot position was (0.96+/-0.27) mm in IEF direction, and (1.24+/-0.41) mm in SDS-PAGE direction indicating relatively good reproducibility of the protein spots. Fifty-eight proteins were identified, certain proteins were products of oncogenes, and others were involved in cell cycle regulation, and signal transduction. CONCLUSIONS: A reference map of NCI-H446 cells was established,certain proteins were identified by MALDI-TOF-MS and PMF. These data will be useful for establishing human SCLC proteome database.

Amino Acid Sequence↗

PubMed2003

Purification and characterization of Hainantoxin-V, a tetrodotoxin-sensitive sodium channel inhibitor from the venom of the spider Selenocosmia hainana.

A neurotoxic peptide, named Hainantoxin-V (HNTX-V), was isolated from the venom of the Chinese bird spider Selenocosmia hainana. The complete amino acid sequence of HNTX-V has been determined by Edman degradation and found to contain 35 amino acid residues with three disulfide bonds. Under whole-cell patch-clamp mode, HNTX-V was proved to inhibit the tetrodotoxin-sensitive (TTX-S) sodium currents while it had no any effects on tetrodotoxin-resistant (TTX-R) sodium currents on adult rat dorsal root ganglion neurons. The inhibition of TTX-S sodium currents by HNTX-V was tested to be concentrate-dependent with the IC(50) value of 42.3nM. It did not affect the activation and inactivation kinetics of currents and did not have the effect on the active threshold of sodium channels and the voltage of peak inward currents. However, 100nM HNTX-V caused a 7.7mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation. The results indicated that HNTX-V inhibited mammalian voltage-gated sodium channels through a novel mechanism distinct from other spider toxins such as delta-ACTXs, micro -agatoxins I-VI which bind to receptor site three to slow the inactivation kinetics of sodium currents.

Amino Acid Sequence↗

PubMed2003

Purification and characterization of raventoxin-I and raventoxin-III, two neurotoxic peptides from the venom of the spider Macrothele raveni.

The spider Macrothele raveni was recently identified as a new species of Genus Macrothele. The crude venom from M. raveni was found to be neurotoxic to mice and the LD(50) of the crude venom in mice was 2.852mg/kg. Two neurotoxic peptides, raventoxin-I and raventoxin-III, were isolated from the crude venom by ion-exchange and reverse phase high performance liquid chromatography. Raventoxin-I was the most abundant toxic component in the venom, while raventoxin-III was a lower abundant component. Both toxins can kill mice and block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation, but have no effect on cockroaches. The LD(50) of raventoxin-I in mice is 0.772mg/kg. The complete amino acid sequences of raventoxin-I and raventoxin-III were determined and found to consist of 43 and 29 amino acid residues, respectively. It was determined by mass spectrometry that all Cys residues from raventoxin-I and raventoxin-III are involved in disulphide bonds. raventoxin-III showed no significant sequence homology with any presently known neurotoxins in the protein/DNA databases, while raventoxin-I has limited sequence identity with delta-AcTx-Hv1 and delta-AcTx-Ar1, which target both mammalian and insect sodium channels. Both raventoxin-I and raventoxin-III only work on vertebrates, but not on insects. Moreover, raventoxin-I could exert an effect of first exciting and then inhibiting the contraction of mouse diaphragm muscle caused by electrically stimulating the phrenic nerve, but raventoxin-III could not.

Amino Acid Sequence↗

PubMed2003

Huwentoxin-V, a novel insecticidal peptide toxin from the spider Selenocosmia huwena, and a natural mutant of the toxin: indicates the key amino acid residues related to the biological activity.

A neurotoxin peptide (named Huwentoxin-V) was purified from the venom of the Chinese bird spider Selenocosmia huwena by a combination of ion exchange chromatography and reverse phase HPLC. HWTX-V has 35 amino acid residues, and is in perfect agreement with the molecular mass 4111.4 Da identified by mass spectrometry. A natural mutant of the toxin (called mHuwentoxin-V) was also isolated from the venom. mHWTX-V was only truncated two amino acid residues from the C-terminus of HWTX-V, and its molecular weight is 3877.1 Da determined by mass spectrometry. The six cysteine residues in each sequence of the two peptides suggest three disulfide bridges, the present of which was demonstrated by mass spectrometry after dithiothreiotol reduce and S-carboxymethylation. The primary structure of the two toxins exhibits sequence identity with other spider toxins such as ProTx-I (64%), SGTx (57%), SNX-482 (55%), and Hanatoxin (54%). HWTX-V can reversibly paralyze locusts and cockroaches for several hours with a ED50 value as 16 +/- 5 microg/g to locusts, and a larger dose of the toxin can cause death. However, mHWTX-V shows no significant effect on locusts and cockroaches. The structure-activity relationship indicates that the residues Phe34 and Ser35 in the C-terminus of HWTX-V are the key residues of the biological activity.

Amino Acid Sequence↗

PubMed2003

Inhibition of sodium channels in rat dorsal root ganglion neurons by Hainantoxin-IV, a novel spider toxin.

The effects of Hainantoxin-IV (HNTX-IV), a neurotoxic peptide isolated from the venom of the Chinese bird spider Seleconosmia hainana, on the adult rat dorsal root ganglion (DRG) neurons were investigated. Using the whole-cell patch-clamp technique HNTX-IV inhibited mammal neural TTX-sensitive (TTX-S) sodium currents evidently but the toxin failed to affect TTX-resistant (TTX-R) ones. The inhibition of HNTX-IV is dose-dependent with the IC(50) value of 44.6 nmol/L. The toxin didn't affect the activation and inactivation kinetics of sodium currents, but it caused a 10.1 mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation on DRG neurons. The results indicated that HNTX-IV, a novel spider toxin, maybe alternate voltage-gated sodium channels through a mechanism distinct from other spider toxins such as delta-ACTXs, mu-agatoxins I-VI which targeted the receptor site 3 to slow the inactivation kinetics of sodium currents.

Animals↗

PubMed2003

[Identification of protein spots in silver-stained two-dimensional gels based on chemically assisted fragmentation by MALDI-TOF-PSD mass spectrometry].

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides,accomplished with O-methylisourea hydrogen sulfate, converted lysine into homoarginine residues, improving detection in MALDI-MS. Then tryptic peptides were labeled with sulfonic acid groups at the N-termini using 3-sulfopropionic acid NHS-ester chemistry. The derivatives enhanced post-source decay (PSD) fragmentation signals and produced a spectrum containing only y-ions. This facilitated de novo peptide sequencing, so it is an important contribution to unambiguous protein identification in proteome research. This method was successfully applied in nasopharyngeal carcinoma(NPC) proteome study.

Amino Acid Sequence↗

PubMed2003

[Researching a novel NPC-related candidate suppressor gene BRD7 by two-dimensional gel electrophoresis and MALDI-TOF-MS].

BRD7 was isolated through cDNA representational difference analysis (RDA) (GenBank accession No. AF152604). Previous studies showed that BRD7 gene was down-regulated in nasopharyngeal carcinoma (NPC) cells and tissues, and three cSNPs (coding-region single nucleotide polymorphisms) were found on BRD7. In addition, six BRD7-interacting proteins were identified by yeast two-hybrid system. To study the function of this gene on carcinogenesis in NPC, BRD7 gene was transfected into HNE1 cells low-expressed BRD7 by using liposomes and a stable cell line over-expressing BRD7 was established. After two-dimensional gel electrophoresis(2-DE), twenty differentially expressed proteins were identified by MALDI-TOF-MS including argininosuccinate lyase, metalloproteinase inhibitor-2 precursor, proteaseome activator28 beta subunit, thyroid transcriptional factor-1, cyclinH (MO15-associated protein), and so on. These differentially expressed proteins are related to cell cycling, transcription regulation, signaling pathway etc. Therefore, BRD7 may exert its functions by mediating differential expression of these proteins.

Carrier Proteins↗

PubMed2003

Purification and characterization of a neurotoxic peptide huwentoxin-III and a natural inactive mutant from the venom of the spider Selenocosmia huwena Wang (Ornithoctonus huwena Wang).

A novel neurotoxic peptide, named huwentoxin-III, and a natural mutant have been isolated from the venom of the spider Selenocosmia huwena Wang (Ornithoctonus huwena Wang). The average molecular weights of the two peptides were determined as 3853.35 and 3667.40 by mass spectrometry, respectively. Huwentoxin-III has 33 amino acid residues containing 6 cysteine residues. Its natural mutant is only truncated a tryptophan residue from C-terminals of huwentoxin-III. The sequences of the two peptides show 70.5% sequence similarity with that of lectin-like peptide SHL-I previously isolated from the venom of the same spider, while they cannot agglutinate human erythrocytes. Huwentoxin-III can reversibly paralyze cockroaches for several hours with an ED(50) of (192.95 +/- 120.84) microg/g (P=0.95) (mean +/- SD) and can enhance the muscular contractions elicited by stimulating the nerve of the isolated rat vas deferens, however, the mutant of huwentoxin-III has no such effect, which suggests that Trp33 was an important residue related to the biological function of huwentoxin-III.

Amino Acid Sequence↗

PubMed2002

The structure of spider toxin huwentoxin-II with unique disulfide linkage: evidence for structural evolution.

The three-dimensional structure of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of spider Selenocosmia huwena with a unique disulfide bond linkage as I-III, II-V, and IV-VI, has been determined using 2D (1)H-NMR. The resulting structure of HWTX-II contains two beta-turns (C4-S7 and K24-W27) and a double-stranded antiparallel beta-sheet (W27-C29 and C34-K36). Although the C-terminal double-stranded beta-sheet cross-linked by two disulfide bonds (II-V and IV-VI in HWTX-II, II-V and III-VI in the ICK molecules) is conserved both in HWTX-II and the ICK molecules, the structure of HWTX-II is unexpected absence of the cystine knot because of its unique disulfide linkage. It suggests that HWTX-II adopts a novel scaffold different from the ICK motif that is adopted by all other spider toxin structures elucidated thus far. Furthermore, the structure of HWTX-II, which conforms to the disulfide-directed beta-hairpin (DDH) motif, not only supports the hypothesis that the ICK is a minor elaboration of the more ancestral DDH motif but also suggests that HWTX-II may have evolved from the same structural ancestor.

Amino Acid Sequence↗

PubMed2002

[Differential proteomic analysis of human lung adenocarcinoma cell line A-549 and of normal cell line HBE].

To explore the differential proteomic expressions between human lung adenocarcinoma cell line A-549 and normal cell line HBE, a series of methods, including immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, silver staining, PDQuest 2-DE software analysis, peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, were used to separate and identify the differential proteomic expressions between A-549 and HBE. The results showed that the good 2-DE pattern including high resolution and reproducibility was obtained. After silver staining, the 2-DE image analysis by PDQuest 2-DE software detected average (890 +/- 38) spots in A-549, and (757 +/- 27) spots in HBE. The average positional deviation of the matched spots between A-549 and HBE 2-DE maps was (2.85 +/- 0.48) mm in IEF direction, and (2.69 +/- 0.37) mm in SDS-PAGE direction. The differential proteomic expression analysis found that there were 535 matched spots between A-549 and HBE 2-DE maps, 355 spots that were not matched in A-549, 222 spots that were not matched in HBE. 18 differential spots (8 spots in A-549 and 10 spots in HBE) were cut off from silver staining gel at random, digested in gel with TPCK-trypsin, measured with MALDI-TOF-MS and searched in the SWISS-PROT database with PeptIdent software. 18 protein were preliminarily identified. These proteins were related to cell signal transduction, cell metabolism, proliferation and differentiation etc. There was a significant difference at protein level between human lung adenocarcinoma cell line A-549 and normal cell line HBE. It suggests that the differential expression analysis of proteomes may be useful to further study of the related proteins and the molecular markers of lung adenocarcinoma.

Adenocarcinoma↗

PubMed2002

Synthesis and oxidative refolding of hainantoxin-IV.

Hainantoxin-IV, a neutoxic peptide from the spider Selenocosimia hainana, was synthesized by solid-phase method with fluorenylmethyoxycarbonyl amino acids (Fmoc-AA). Reverse-phase HPLC was used to monitor the oxidative folding of synthetic Hainantoxin-IV under different reaction conditions in order to find optimal conditions for renaturation of synthetic Hainantoxin-IV. The best renaturation yield was received in 5 mmol/L GSH and 0.5 mmol/L GSSG at pH 8.0 in 0.1 mol/L Tris-HCl and 0.1 mol/L NaCl buffer. The renaturated Hainantoxin-IV was monitored with MALDI-TOF MS reverse-phase HPLC and isolated mouse phrenic nerve-diaphragm preparation.

Amino Acid Sequence↗