A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor,;encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization. (C) 2004 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.

Molecular cloning of TRAF2 binding protein gene and its promoter region from the grass carp Ctenopharyngodon idellus

A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappa B (NF kappa B) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection. (c) 2005 Elsevier B.V. All rights reserved.

Chitosan(Chitin)/Cellulose Composite Biosorbents Prepared Using Ionic Liquid for Heavy Metal Ions Adsorption

Chitosan(chitin)/cellulose composites as biodegradable biosorbents were prepared under an environment-friendly preparation processes using ionic liquids. Infrared and X-ray photoelectron spectra indicated the stronger intermolecular hydrogen bond between chitosan and cellulose, and the hydroxyl and amine groups were believed to be the metal ion binding sites. Among the prepared biosorbents, freeze-dried composite had higher adsorption capacity and better stability. The capacity of adsorption was found to be Cu(II) (0.417 mmol/g) > Zn(II) (0.303 mmol/g) > Cr(VI) (0.251 mmol/g) > Ni(II) (0.225 mmol/g) > Ph(II) (0.127 mmol/g) at the same initial concentration 5 mmol L-1. In contrast to some other chitosan-type biosorbents, preparation and component of the biosorbent were obviously more environment friendly. Moreover, adsorption capacity of chitosan in the blending biosorbent could be fully shown.

Diffusion and single molecule dynamics on biomolecular interface binding energy landscape

We propose a new approach to study the diffusion dynamics on biomolecular interface binding energy landscape. The resulting mean first passage time (MFPT) has 'U'curve dependence on the temperature. It is shown that the large specificity ratio of gap to roughness of the underlying binding energy landscape not only guarantees the thermodynamic stability and the specificity [P.A. Rejto, G.M. Verkhivker, in: Proc. Natl. Acad. Sci. 93 (1996) 8945; C.J. Tsai, S. Kumar, B. Ma, R. Nussinov, Protein Sci. 8 (1999) 1181; G.A. Papoian, P.G. Wolynes, Biopolymers 68 (2003) 333; J. Wang, G.M. Verkhivker, Phys. Rev. Lett. 90 (2003) 198101] but also the kinetic accessibility. The complex kinetics and the associated fluctuations reflecting the structures of the binding energy landscape emerge upon temperature changes. The theory suggests a way of connecting the models/simulations with single molecule experiments by analysing the kinetic trajectories.

The interaction of aldolase with the cytoplasmic domain of human erythrocyte band 3 inhibited by lanthanum ions

The effect of lanthanum ions on the activity of the cytoplasmic domain of human erythrocyte band 3 (CDB3), which was measured according to the inhibition to aldolase, was studied. In the presence of low concentration of lanthanum ions, the function of CDB3 to inhibit aldolase activity decreased significantly. It indicated that lanthanum ions in the erythrocyte would change the conformation of CDB3 and influence the control on aldolase activity.

Study on polymer micelles of hydrophobically modified ethyl hydroxyethyl cellulose using single-molecule force spectroscopy

Individual hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC) molecules under different conditions were elongated using a new atomic force microscope (AFM) based technique-single-molecule force spectroscopy (SMFS). The critical concentration of HM-EHEC for micelle-like clusters at a solid/liquid interface was around 0.8 wt %, which is lower than that in solution. The different mechanical properties of HM-EHEC below and above the critical concentration were displayed on force-extension curves. Through a comparison with unmodified hydroxyethyl cellulose, substituent-induced effects on nanomechanical features of HM-EHEC were investigated. Because of hydrophobic interactions and cooperative binding with the polymer, surfactants such as sodium dodecyl sulfate (SDS) dramatically influence the elastic properties of HM-EHEC below the critical concentration, and further addition of SDS reduces the interactions between the hydrophobic groups and the surfactant.

Domain analysis of the Edwardsiella tarda ferric uptake regulator

Recent studies have shown that the ferric uptake regulator (Fur) of Edwardsiella tarda (Fur(Et)) shares high sequence identity with the Escherichia coli Fur (Fur(Ec)) at the N-terminal DNA-binding region. In the present study, the functional importance of the C-terminal region of Fur(Et) was investigated. It was found that Fur(Et) bearing deletion of the C-terminal 12 residues still possesses most of the repressor activity, whereas Fur(Et) bearing deletions of the C-terminal 16 and more than 16 residues are severely affected in activity. Domain swapping analyses indicated that the chimeric Fur proteins (Et75Ec73 and Et75Vh74) consisting of the N-terminal 1-75 region of Fur(Et) fused to the C-terminal 76-148 region of Fur(Ec) and the C-terminal 76-149 region of the Vibrio harveyi Fur (Fur(Vh)), respectively, are fully active. C92 of Fur(Ec) and C137 of Fur(Vh), which are functionally essential in Fur(Ec) and Fur(Vh), respectively, are also essential in Et75Ec73 and Et75074, respectively. Further study identified an artificial Fur protein, EtMF54, which is composed of the N-terminal 49 residues of Fur(Et) and five artificial residues. Compared to Fur(Et), EtMF54 possesses partial Fur activity that is iron-dependent. These results (I) indicate that there exist certain functional/structural compatibilities among Fur(Et), Fur(Ec), and Fur(Vh) at the C-terminal region; (ii) provide insights to the potential location of the regulatory ion-binding site of Fur(Et).

SmC2P1, a C2 domain protein from Scophthalmus maximus that binds bacterial pathogen-infected lymphocytes and reduces bacterial survival

C2 domains are protein structural modules found in many eukaryotic proteins involved in signal transduction, membrane trafficking, and immune defense. Most of the studied C2 domain-containing proteins are multi-domained in structure, in which the C2 domain is an independently folded motif and plays an essential role in calcium-dependent membrane-targeting. Although C2 domains isolated from intact proteins have been studied for biological functions, no study on natural proteins containing C2 domain only has been documented. In this study, we identified a Scophthalmus maximus protein SmC2P1 that is comprised of a single C2 domain and lacks any other apparent domain structures. The deduced amino acid sequence of SmC2P1 contains 129 residues and shares 36-38% identities with the C2 domains of the perforins of several fish species. Like typical C2 domains, SmC2P1 is predicted to organize into eight beta-strands with a Ca2+-binding site located in inter-strand loops. SmC2P1 expression was detected, in deceasing order, in liver, spleen, blood, brain, muscle, kidney, gill, and heart. Experimental challenge of turbot with a bacterial pathogen significantly upregulated SmC2P1 expression in kidney in a time-dependent manner. Recombinant SmC2P1 purified from yeast exhibits no hemolytic activity but binds to pathogen-infected kidney lymphocytes in the presence of calcium. Furthermore, interaction of recombinant SmC2P1 with bacterium-infected lymphocytes reduced bacterial survival. These results indicate that SmC2P1 is a functional protein that is involved in host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.