Isolation and characterization of two Korean mistletoe lectins

Two isolectins (KML-IIU and the KML-IIL) were individually isolated from the previously reported Korean mistletoe lectin, KML-C, by using an immunoaffinity column. Molecular weights of the KML-IIU and the KML-IIL were 64 kDa and 60 kDa respectively. Both of the lectins were composed of heterogeneous A and B subunits linked with a disulfide bond, and showed the same carbohydrate-binding specificities for Gal and GalNAc. However, they are different not only in biophysical properties (glycosylation and amino acid compositions) but also bioactivities (cell killing and cytokine induction). The KML-IIL showed 17-145 times stronger in cytotoxicities to various human and mouse cancer cell lines than the KML-IIU. The KML-IIL also induced TNF-alpha secretion from mouse peritoneal macrophages 4.5 times better than the KML-IIU. The results demonstrated isolectins in Korean mistletoe were varied in bioactivities and the KML-IIL may be developed as an anti-cancer agent.     

Genomic distribution of simple sequence repeats in Brassica rapa

Simple Sequence Repeats (SSRs) represent short tandem duplications found within all eukaryotic organisms. To examine the distribution of SSRs in the genome of Brassica rapa ssp. pekinensis, SSRs from different genomic regions representing 17.7 Mb of genomic sequence were surveyed. SSRs appear more abundant in non-coding regions (86.6%) than in coding regions (13.4%). Comparison of SSR densities in different genomic regions demonstrated that SSR density was greatest within the 5'-flanking regions of the predicted genes. The proportion of different repeat motifs varied between genomic regions, with trinucleotide SSRs more prevalent in predicted coding regions, reflecting the codon structure in these regions. SSRs were also preferentially associated with gene-rich regions, with peri-centromeric heterochromatin SSRs mostly associated with retrotransposons. These results indicate that the distribution of SSRs in the genome is non-random. Comparison of SSR abundance between B. rapa and the closely related species Arabidopsis thaliana suggests a greater abundance of SSRs in B. rapa, which may be due to the proposed genome triplication. Our results provide a comprehensive view of SSR genomic distribution and evolution in Brassica for comparison with the sequenced genomes of A. thaliana and Oryza sativa.     

Analysis of active center in hyperthermophilic cellulase from Pyrococcus horikoshii

A hyperthermostable endoglucanase from Pyrococcus horikoshii with the capability of hydrolyzing crystalline cellulose was analyzed. A protein engineering study was carried out to obtain a reduced-size mutant. Five amino acid residues at both the N- and C-terminus were found to be removable without any loss of activity or thermal stability. Site-directed mutagenesis was also performed on R102, N200, E201, H297, Y299, E342, and W377, residues possibly involved in the active center or in the recognition and binding of a cellulose substrate. The activity of the resulting mutants was considerably decreased, confirming that the mutated residues were all important for activity. A reduced-size enzyme, as active as the wild-type endoglucanase, was successfully obtained, plus the residues critical for its activity and specificity were confirmed. Consequently, an engineered enzyme with a reduced size was obtained, and the amino acids essential for activity were confirmed by site-directed mutagenesis and comparison with a known three-dimensional structure.     

Development of a protein secretion system with the application of sec-dependent protein secretion components

In order to induce high levels of protein secretion, we have constructed a recombinant plasmid, designated pBP244, into which was incorporated key components of the type-II Sec-dependent secretion system, including LepB (signal peptidase), SecA (ATPase), and SecB (chaperone). The biological activities of the LepB, SecA, and SecB components expressed from genes harbored by pBP244 appeared to play their normal roles. In order to evaluate the protein secretion, a pspA (Streptococcus pneumoniae surface protein A) gene was cloned into pBP244, resulting in pBP438. S. typhimurium harboring pBP438 grown until the stationary phase, secreted a higher level of PspA into the culture supernatants than did the strain harboring pYA3494. The strain harboring pBP438 secreted a supernatant amount 1.71-fold, a periplasmic space amount 1.47-fold, and an outer membrane amount 1.49-fold higher than that of pYA3494. S. typhimurium chi8554 kept the Asd+ plasmid pBP244 and pBP438 for 60 generations in LB broth harboring DAP, thereby indicating that pBP244 and pBP438 were quite stable in the Salmonella strain.     

Transcription profile in mouse four-cell, morula, and blastocyst: Genes implicated in compaction and blastocoel formation

To gain insight into early embryo development, we utilized microarray technology to compare gene expression profiles in four-cell (4C), morula (MO), and blastocyst (BL) stage embryos. Differences in spot intensities were normalized, and grouped by using Avadis Prophetic software platform (version 3.3, Strand Genomics Ltd.) and categories were based on the PANTHER and gene ontology (GO) classification system. This technique identified 622 of 7,927 genes as being more highly expressed in MO when compared to 4C (P < 0.05); similarly, we identified 654 of 9,299 genes as being more highly expressed in BL than in MO (P < 0.05). Upregulation of genes for cytoskeletal, cell adhesion, and cell junction proteins were identified in the MO as compared to the 4C stage embryos, this means they could be involved in the cell compaction necessary for the development to the MO. Genes thought to be involved in ion channels, membrane traffic, transfer/carrier proteins, and lipid metabolism were also identified as being expressed at a higher level in the BL stage embryos than in the MO. Real-time RT-PCR was performed to confirm differential expression of selected genes. The identification of the genes being expressed in here will provide insight into the complex gene regulatory networks effecting compaction and blastocoel formation.     

Gene delivery properties of end-modified poly(beta-amino ester)s

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.     

One step engineering of T7-expression strains for protein production: increasing the host-range of the T7-expression system

The T7-expression system has been very useful for protein expression in Escherichia coli. However, it is often desirable to over-express proteins in species other than E. coli. Here, we constructed an inducible broad-host-range T7-expression transposon, which allows simple one-step construction of T7-expression strains in various species, providing the option to over-express proteins of interest in a broader host-range. This transposon contains the T7 RNA polymerase driven by the lacUV5 promoter, which is repressed by the lac-repressor. Leaky expression is prevented by the presence of T7-lysozyme on this construct. The complete T7-expression system is flanked by mariner transposon repeats of the suicidal R6Kgammaori plasmid, pBT20-Deltabla. Stable integration of the whole system is possible by a one-step selection for a Flp-excisable Gm(R)-marker. We showed the engineering of E. coli, Pseudomonas aeruginosa, Erwinia carotovora, Salmonella choleraesuis, Agrobacterium tumefaciens, and Chromobacterium violaceum strains with this construct and demonstrated the expression of the Burkholderia pseudomallei Asd protein in these hosts, by induction with isopropyl-beta-d-thiogalactopyranoside (IPTG).     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.