Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis, with emphasis on origin of European domestic carp.

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5-ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5-ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo.

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%).

Rational Redesign of Inhibitors of Furin/kexin Processing Proteases.

Furin/kexin processing proteases catalyze the proteolysis of large protein precursors involved in many biological processes, such as zymogen activation, peptide hormone synthesis, viral protein processing and receptor maturation, making them potential targets for therapeutic agents. Herein, homology modeling and weighted evolutionary tracing were combined to investigate the interactionmechanism of furin/kex2 with eglin C mutants. The model structures showed that there were many acidic residues in the furin (kex2) binding interface, contributing to specificity for multiple basic residues of their corresponding substrates or inhibitors. Besides, some rational explanations were presented for the different inhibitor/substrate specificity of the furin/kexin members by combining the model structures with results of evolutionary tracing. Based on these analyses,an attempt was made to rationally redesign the eglin C by interface engineering with heterogeneous self-consistent ensemble optimization to improve its inhibitory specificity on furin/kex2. With the model complex structures of furin/kex2 and eglin C variants as structural templates, the P(1), P(2) and P(4) of eglin C were redesigned, respectively. The design results show that both furin and kex2 favored basic residues at P(1), P(2) and P(4) in eglin C, in good agreement with the experimental data. The detection of many specific residues in S' part of furin/kexin sequences made possible designing inhibitors with high specific binding to furin and kex2, respectively. As for furin, the best inhibitor designed was eglin C-P(2)'Glu-P(3)'Asp-P(4)'Arg (only these three positions were shown), while the best eglin C variant for kex2 designed was P(2)'Arg-P(3)'Arg-P(4)'Glu. The structures show that furin and kex2 form distinct interactions with these two eglin C variants. Herein, a strategy was proposed that combine homology modeling, evolutionary tracing and rational interface redesign to investigate enzyme-inhibitor interactions and inhibitor engineering. This computational design gives some rational guidance to further experimental inhibitor engineering.

Enhancing Penicillin G Acylase Stability by Site-directed Mutagenesis.

To improve the stability of penicillin G acylase(PGA) from Bacillus megaterium, a three-dimensional model of B.megaterium PGA was constructed based on crystal structure of penicillin G acylase from E.coli using PMODELING program. The mutation of Lys at beta427 and 430 to Ala was predicted to enhance the stability of PGA in acidic or organic solvent environment. The results showed that 2 mutant PGA had similar specific activity and Km as the parent PGA. Their optimum pH dropped 0.5 pH units. The stability of Lys430Ala was enhanced obviously at pH 5.2. The half lives of Lys427Ala and Lys430Ala were improved by 60 % and 166 %, respectively, in comparison with the parent PGA.

Engineering Novel Functional Proteins Grafting Active Sites into Natural Scaffolds.

Engineering novel small functional proteins by grafting active sites into small but stable proteins is an efficient protein design method. Combining heterogeneous self-consistent ensemble optimization (hetero-SCEO) with 3D-motif search tool, we developed a system to accomplish such method. It is tested by transferring zinc-binding site of carbonic anhydrase form B to charybdotoxin and its efficiency is demonstrated.

Flexible Docking of Proteins and "Drug-like" Ligands.

By using "tabu search" algorithm and Gehlhaar potential function, a new approach is presented for flexible docking of protein and its "drug-like" ligand has been developed. Computational test for this method with a set of 100 complexes has been performed, which indicated that the deviation of 89% of the predicted complex conformation was less than 0.25 nm. Compared with GOLD, a program of genetic algorithm, our method has high accuracy, low limit and short computation time.

Design of Protein Cores by Screening Combinatorial Sequence Library.

We have developed a new method, heterogeneous self-consistent ensemble optimization (hetero-SCEO), to select appropriate hydrophobic cores of proteins. It has been tested with five kinds of proteins: lambda-repressor, phage 434 CRO protein, interleukin-4, thioredoxin and ubiquitin. The results show that the method can be used for the de novo desigh of the hydrophobic cores of proteins.