Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Docking and molecular dynamics studies toward the binding of new natural phenolic marine inhibitors and aldose reductase

Phenolic marine natural product is a kind of new potential aldose reductase inhibitors (ARIs). In order to investigate the binding mode and inhibition mechanism, molecular docking and dynamics studies were performed to explore the interactions of six phenolic inhibitors with human aldose reductase (hALR2). Considering physiological environment, all the neutral and other two ionized states of each phenolic inhibitor were adopted in the simulation. The calculations indicate that all the inhibitors are able to form stable hydrogen bonds with the hALR2 active pocket which is mainly constructed by residues TYR48, HIS110 and TRP111, and they impose the inhibition effect by occupying the active space. In all inhibitors, only La and its two ionized derivatives La_ion1 and La_ion2, in which neither of the ortho-hydrogens of 3-hydroxyl is substituted by Br, bind with hALR2 active residues using the terminal 3-hydroxyl. While, all the other inhibitors, at least one of whose ortho-sites of 3- and 6-hydroxyls are substituted by Br substituent which take much electron-withdrawing effect and steric hindrance, bind with hALR2 through the lactone group. This means that the Br substituent can effectively regulate the binding modes of phenolic inhibitors. Although the lactone bound inhibitors have relatively high RMSD values, our dynamics study shows that both binding modes are of high stability. For each inhibitor molecule, the ionization does not change its original binding mode, but it does gradually increase the binding free energy, which reveals that besides hydrogen bonds, the electrostatic effect is also important to the inhibitor–hALR2 interaction.

Determination of total iron binding capacity of serum by capillary electrophoresis

A capillary electrophoresis (CE) technique for determining total iron binding capacity (TIBC) of serum has been developed. The optimum serum pretreatment involves the following major steps: at first, saturate serum transferrin with Fe+3; then, dissociate them completely after removing excess unbound Fe. Finally, complex the released iron with phenanthroline, a chromophore, to make suitable for the CE analysis. Ammonium acetate (pH = 5.0) was used as CE background electrolyte solution. In this system, a good linear correlation coefficient was maintained over the range 0.5 similar to 10 mu M (r = 0.9979, n =12). Seven adult serum samples were studied and the TIBC parameters measured. In the present system, 10 similar to 30 mu L serum is sufficient for determination. The study shows that the CE technique described is a powerful method for rapid, efficient, sensitive and reliable analysis and hence particularly suitable for clinical application.

Capillary electrophoresis study of human serum albumin binding to basic drugs

The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm x 50 mu m i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients, r > 0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.

Capillary zone electrophoresis characterization of low molecular weight heparin binding to interleukin 2

A method based on capillary zone electrophoresis (CZE) was used to study the interaction between low molecular weight heparin (LMWH) and interleukin 2 (IL-2). The results showed that the increase of the concentration of LMWH led to the decrease of the peak height and the increase of the peak width of IL-2, but the peak areas were kept constant. The binding constant of IL-2 with LMWH was calculated as 1.2 x 10(6) M(-1) by Scatchard analysis, which is in good agreement with the results found in the references using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the interaction between IL-2 and LMWH is of fast on-and-off kinetic binding reaction. CZE might be used to study not only slow on-and-off rates interactions, but also fast on-and-off rates ones. The binding constant can be calculated easily, and the method can be applied to study a wide range of heparin-protein interactions. (c) 2005 Elsevier B.V. All rights reserved.