Bombinakinin M gene associated peptide, a novel bioactive peptide from skin secretions of the toad Bombina maxima

A novel 28-amino acid peptide, termed bombinakinin-GAP, was purified and characterized from skin secretions of the toad Bombina maxima. Its primary structure was established as DMYEIKQYKTAHGRPPICAPGEQCPIWV-NH2, in which two cysteines form a disulfide bond. A FASTA search of SWISS-PROT databank detected a 32% sequence identity between the sequences of the peptide and a segment of rat cocaine- and amphetamine-regulated transcript (CART). Intracerebroventricular (i.c.v.) administration of the peptide induced a significant decrease in food intake in rats, suggesting that it played a role in the control of feeding by brain. Analysis of its cDNA structure revealed that this peptide is coexpressed with bombinakinin M, a bradykinin-related peptide from the same toad. Bombinakinin-GAP appears to be the first example of a novel class of bioactive peptides from amphibian skin, which may be implicated in feeding behavior. (C) 2003 Elsevier Science Inc. All rights reserved.

Cloning of novel bombesin precursor cDNAs from skin of Bombina maxima

Amphibian skin is a rich resource of bioactive peptides like proline-rich bombesin from frog Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises proline-rich bombesin and a novel peptide, designated as bombestatin, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of the novel peptide is WEVLLNVALIRLELLSCRSSKDQDQKESCGMHSW, in which two cysteines form a disulfide bond. A BLAST search of databases did not detect sequences with significant similarity. Bombestatin possesses dose-dependent contractile activity on rat stomach strips. The differences between cDNAs encoding PR-bombesin plus bombestatin and PR-bombesin alone are due to fragment insertions located in 3'-coding region and 3'-untranslational region, respectively. (c) 2005 Elsevier B.V. All rights reserved.

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B. maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1: 1 molar ratio. The equilibrium dissociation constants (K-D) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys(53)-Cys(62)), which comprises the scissile bond Arg(58)(P-1)-His(59)(P-1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

A new protein structure of P-II class snake venom metalloproteinases: it comprises metalloproteinase and disintegrin domains

A new metalloproteinase-disintegrin, named Jerdonitin, was purified from Trimeresurus jerdonii venom with a molecular weight of 36 kDa on SDS-PAGE. It dose-dependently inhibited ADP-induced human platelet aggregation with IC50 of 120 nM. cDNA cloning and sequencing revealed that Jerdonitin belonged to the class II of snake venom metalloproteinases (SVMPs) (P-II class). Different from other P-II class SVMPs, metalloproteinase and disintegrin domains of its natural protein were not separated, confirmed by internal peptide sequencing. Compared to other P-II class SVMPs, Jerdonitin has two additional cysteines (Cys219 and Cys238) located in the spacer domain and disintegrin domain, respectively. They probably form a disulfide bond and therefore the metalloproteinase and disintegrin domains cannot be separated by posttranslationally processing. In summary, comparison of the amino acid sequences of Jerdonitin with those of other P-II class SVMPs by sequence alignment and phylogenetic analysis, in conjunction with natural protein structure data, suggested that it was a new type of P-II class SVMPs. (C) 2003 Elsevier Inc. All rights reserved.

Characterize dynamic conformational space of human CCR5 extracellular domain by molecular modeling and molecular dynamics simulation

The chemokine receptor CCR5 is the receptor for several chemokines and major coreceptor for R5 human immunodeficiency virus type-1 strains entry into cell. Three-dimensional models of CCR5 were built by using homology modeling approach and 1 ns molecular dynamics (MD) simulation, because studies of site-directed mutagenesis and chimeric receptors have indicated that the N-terminus (Nt) and extracellular loops (ECLs) of CCR5 are important for ligands binding and viral fusion and entry, special attention was focused on disulfide bond function, conformational flexibility, hydrogen bonding, electrostatic interactions, and solvent-accessible surface area of Nt and ECLs of this protein part. We found that the extracellular segments of CCR5 formed a well-packet globular domain with complex interactions occurred between them in a majority of time of MID simulation, but Nt region could protrude from this domain sometimes. The disulfide bond Cys20-Cys269 is essential in controlling specific orientation of Nt region and maintaining conformational integrity of extracellular domain. RMS comparison analysis between conformers revealed the ECL1 of CCR5 stays relative rigid, whereas the ECL2 and Nt are rather flexible. Solvent-accessible surface area calculations indicated that the charged residues within Nt and ECL2 are often exposed to solvent. Integrating these results with available experimental data, a two-step gp120-CCR5 binding mechanism was proposed. The dynamic interaction of CCR5 extracellular domain with gp120 was emphasized. (C) 2004 Elsevier B.V. All rights reserved.

含二硫键多肽的基质辅助激光解析电离飞行时间质谱研究

The determination of disulfide bonds becomes an important aspect of obtaining a comprehensive understanding of the chemical structure of a protein. Numerous experimental methods have been developed for the determination of disulfide bonds in proteins. Modern mass spectrometry has developed as an important tool for the analysis of disulfide bond patterns due to its advantages of being simple, rapid and sensitive. The dissociations of the disulfide bonds were detected during the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. These fragment ions were attributed to prompt fragmentation or “in-source decay” rather than “post-source decay”. For the double disulfide bonds, ions of plus sulfur and minus sulfur atoms corresponding to cleavages at different sites within the carbon-sulfur-sulfur-carbon disulfide bonds were also observed.

SYNTHESIS AND CHARACTERIZATION OF A CROSSLINKING POLYETHYLENIMINE AS SMART GENE CARRIER AND EFFECTS OF PEGYLATION DEGREE

A smart biodegradable cationic polymer (CBA-PEI) based on the disulfide bond-containing cross-linker cystamine bisacrylamide (CBA) and low molecular weight branched polyethylenimine (1800-Da, PEI1800) was successfully synthesized by Michael addition reaction in our recent study. Furthermore, a series of copolymers (CBA-PEI-PEG) with different PEGylation degree were obtained by the mPEG-SPA (5000-Da) reacting with CBA-PEI at various weight ratios directly. The molecular structures of the resulting polymers CBA-PEI and CBA-PEI-PEG were evaluated by nuclear magnetic resonance spectroscopy (H-1-NMR) and capillary viscosity measurements, all of which had successfully verified formation of the copolymers. The polymer/DNA complexes based on CBA-PEI and CBA-PEI-PEG were measured by dynamic light scattering and gel retardation assay. The results showed that the particle size and zeta potential of complexes were reduced with increasing amount of PEG grafting, even no particle formation. The particle size of CBA-PEI/DNA complexes was in range of 103.1 to 129.1 nm, and the zeta potential was in range of 14.2 to 24.3 mV above the 2:1 weight ratio. In the same measure condition, the particle size of CBA-PEI-PEG complexes was reduced to a range of 32.2 to 55 nm, and the zeta potential was in range of 9.3 to 13.8 mV at the 2:1 weight ratio.

Expression of Scophthalmus maximus CD83 correlates with bacterial infection and antigen stimulation

CD83 is a transmembrane glycoprotein of the immunoglobulin (Ig) superfamily and a surface marker for fully matured dendritic cells (DCs) in humans and mice. In teleosts, DC-like cells and their molecular markers are largely unknown. In this report, we described the identification and expressional analysis of a CD83 homologue, SmCD83, from turbot Scophthalmus maximus. The open reading frame of SmCD83 is 639 bp, which is preceded by a S'-untranslated region (UTR) of 87 bp and followed by a 3'-UTR of 1111 bp. The SmCD83 gene is 4716 bp in length, which contains five exons and four introns. The deduced amino acid sequence of SmCD83 shares 40-50% overall identities with the CD83 of several fish species. Like typical CD83, SmCD83 possesses an Ig-like extracellular domain, a transmembrane domain, and a cytoplasmic domain. The conserved disulfide bond-forming cysteine residues and the N-linked glycosylation sites that are preserved in CD83 are also found in SmCD83. Expressional analysis showed that constitutive expression of SmCD83 was high in gill, blood, spleen, muscle, and kidney and low in heart and liver. Bacterial infection and poly(I:C) treatment enhanced SmCD83 expression in kidney in time-dependent manners. Likewise, bacterial challenge caused significant induction of SmCD83 expression in cultured macrophages. Vaccination of turbot with a bacterin and a purified recombinant subunit vaccine-induced significant SmCD83 expression during the first week following vaccination. These results demonstrate that SmCD83 expression correlates with microbial challenge and antigen stimulation, which suggests the possibility that there may exist in turbot DC-like antigen-presenting cells that express SmCD83 upon activation by antigen uptake. In addition, these results also suggest that SmCD83 may serve as a marker for activated macrophages in turbot. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and molecular analysis of a novel C-type lectin from Scophthalmus maximus

C-type lectins are calcium-dependent carbohydrate-binding proteins that play Important roles in innate immunity In this study, a C-type lectin homologue (SmLec1) was identified from turbot (Scophthalmus maximus) and analyzed at expression and functional levels. The open reading frame of SmLec1 is 504 bp, with a 5'-untranslated region (UTR) of 101 bp and a 3'-UTR of 164 bp The deduced amino acid sequence of SmLec1 shares 34%-38% overall identities with the C-type lectins of several fish species In silico analysis identified in SmLec1 conserved C-type lectin features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, and the mannose-type carbohydrate-binding motif In addition, SmLec1 possesses a putative signal peptide sequence and is predicted to be localized in the extracellular. Expression of SmLec1 was highest in liver and responded positively to experimental challenges with fish pathogens Recombinant SmLec1 (rSmLec1) purified from yeast was able to agglutinate the Gram-negative fish pathogen Listonella anguillarum but not the Gram-positive pathogen Streptococcus uncle The agglutinating ability of rSmLec1 was abolished in the presence of mannose and ethylenediaminetetraacetic acid and by elevated temperature (65 degrees C) Further analysis showed that rSmLec1 could stimulate kidney lymphocyte proliferation and enhance the killing of bacterial pathogen by macrophages Taken together, these results suggest that SmLec1 is a unique mannose-binding C-type lectin that possesses apparent immunomodulating property and is likely to be involved in host defense against bacterial infection (C) 2010 Elsevier Ltd. All rights reserved

Redox reaction of disulfide polyaniline in aqueous solution

The cleavage and formation of the di sulfide bond of 2,5-dimercapto-1,3,4-thiadiazole (DMcT) were examined in an aqueous solution of pH value from 0 to 14 with and without polyaniline (PAn), The redox reaction of DMcT was accelerated by PAn in acidic condition. The cell using this anodic material was set-up and characterized in aqueous electrolyte.

Measuring Receptor/Ligand Interaction at the Single-Bond Level: Experimental and Interpretative Issues

There is increased interest in measuring kinetic rates, lifetimes, and rupture forces of single receptor/ligand bonds. Valuable insights have been obtained from previous experiments attempting such measurements. However, it remains difficult to know with sufficient certainty that single bonds were indeed measured. Using exemplifying data, evidence supporting single-bond observation is examined and caveats in the experimental design and data interpretation are identified. Critical issues preventing definitive proof and disproof of single-bond observation include complex binding schemes, multimeric interactions, clustering, and heterogeneous surfaces. It is concluded that no single criterion is sufficient to ensure that single bonds are actually observed. However, a cumulative body of evidence may provide reasonable confidence. 0 2002 Biomedical Engineering Society.

Rayleigh-Taylor Instability of a Liquid Drop at High Bond Numbers

给出了高Bond数下黏性液滴表面Rayleigh-Taylor线性不稳定性的分析解,这种不稳定性对于超音速气流作用下液滴破碎的早期阶段起着至关重要的作用.基于稳定性分析的结果,导出了用于估算稳定液滴的最大直径及液滴无量纲初始破碎时间的计算式,这些计算式与相关文献给出的实验和分析结果比较显示了良好的一致.

Low Spring Constant Regulates P-Selectin-Psgl-1 Bond Rupture

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate r(f) (>= 10(2) pN/s), defined as the product of spring constant k and retract velocity v, how the low r(f) (< 10(2) pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r(f) <= 20 pN/s with low k (similar to 10(-3)-10(-2) pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when r(f) increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at the same r(f). The most probable force, f