Characterization of the hamster genomic fragment cloned by TAR cloning technology with interspecific sequence information

CHO (Chinese Hamster ovary) cells are widely used for biotechnology and biomedical purposes, and now the EST library database of CHO cells is built. Based on this, the construction of the hamster genome library is under exertion. Though the transformation-associated recombination (TAR) cloning method is accounted as an innovative cloning technology without the construction of the genome library in human and mouse, there has been no trial to isolate the genomic fragment from hamster genome by TAR cloning. In this study, approximately 31 kb of hamster genomic fragment was isolated from the normal human/hamster mono-chromosomal somatic cell line (UV5HL9-5B) using universal hooks of rodent repeats sequence of B1 and B2 by TAR cloning. This fragment was analyzed by bioinformatics tools related to the genome alignment for the similarity analysis among rodent and primate, and was classified into rodents by phylogenetic analysis. One putative gene was found in this region which has homology with the human c14orf4 gene. A zinc finger protein domain was found in the translated hamster ORF. Therefore, we suggest that TAR cloning technique can be applied in CHO cells using mouse genomic information, and it can lead to the establishment of the hamster genome database.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Generation of <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> Ghost by Coexpression of PhiX 174 Lysis <Emphasis Type="Italic">E</Emphasis> gene and Staphylococcal Nuclease <Emphasis Type="Italic">A</Emphasis> Gene

Vibrio anguillarum ghosts (VAG) were generated, for the first time, using a conjugation vector containing a ghost bacteria inducing cassette, pRK-λP_R-cI-Elysis, in which the expression of PhiX174 lysis gene E was controlled by the P _R /cI regulatory system of lambda phage. By scanning electron microscopy, holes ranging 80–200 nm in diameter were observed in the VAG. To avoid the presence of bacterial genomic DNA and an antibiotic resistance gene in the final VAG product, we constructed a new dual vector, pRK-λP_R-cI-E-SNA, containing the E-mediated lysis cassette and the staphylococcal nuclease A (SNA)-mediated DNA degradation cassette, and generated safety-enhanced VAG for use as a fish vaccine.     

Directing vanillin production from ferulic acid by increased acetyl-CoA consumption in recombinant Escherichia coli

The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl-CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5alpha (pTAHEF-gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5alpha (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl-CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD-2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli.     

Mapping of Altromycin B-DNA Adduct at Nucleotide Resolution in the Human Genomic DNA by Ligation-mediated PCR

The ligation-mediated PCR was used to map DNA alkylation sites induced by altromycin B at nucleotide resolution in genomic DNA purified from cultured human colon carcinoma. Altromycin B, one of the pluramycin group of antitumor antibiotics, is characterized as intercalator with the added ability to alkylate N7 guanine. DNA adducts formed in genomic DNA were cleaved into DNA strand breaks by hot piperidine treatment, and fragments containing ligatable breaks were then amplified in a single-sided, ligation-mediated PCR. The alkylation sites observed in exon 9 of the p53 gene revealed that the most high reactivity sites for altromycin B were found to be N7 of guanine in a 5-AG* sequence. Determination of the DNA alkylation sites in naked radiolabeled plasmid DNA also showed that altromycin B preferred N7 of guanine in a 5-AG* sequence. Thus, it can be concluded that the sequence selective DNA adduct formation induced by the intercalating alkylator, altromycin B, in genomic DNA is similar to that observed in naked plasmid DNA.     

Comparison of Nitrogen Fixation for North- and South-facing <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> Stands in Central Korea

The nitrogenase activity, root nodule biomass, and rates of nitrogen (N) fixation were measured in 25-year-old pure north- and south-facing Robinia pseudoacacia stands in an urban forest of Seoul (Kkachisan Mountain) in central Korea. The nitrogenase activity was estimated using an acetylene reduction (AR) assay, which showed an increasing trend during the early growing season, with sustained high rates from June through to September with a decrease thereafter. July had the highest nitrogenase activity rate (micromoles C₂H₄ per gram dry nodule per hour), averaging 95.8 and 115.1 for the north- and south-facing stands, respectively. The maximum root nodule biomass (kilograms per hectare) was 45.7 and 9.1 for the north- and south-facing stands in July, respectively. The AR rate appeared to be strongly correlated to the soil temperature (r ² = 0.68, P < 0.001) and soil pH (r ² = 0.59, P < 0.001) while root nodule biomass was correlated to the soil temperature (r ² = 0.36, P < 0.01) and water content (r ² = 0.35, P < 0.05). The soil temperature showed clear differences between seasons, while there was a significant difference in soil pH, organic matter, total N concentrations, and available phosphorus between the north- and south-facing stands. The N₂ fixation rates during the growing season varied from 0.1 to 37.5 kg N ha⁻¹ month⁻¹ depending on the sampling location and time. The annual N₂ fixation rate (kg N per hectare per year) was 112.3 and 23.2 for the north- and south-facing stands, respectively. The differences in N₂ fixation rate between the two stands were due mainly to the differences in total nodule biomass.     

Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia

Autophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic beta cells and glucose homeostasis has not been described. We produced mice with beta cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. beta cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of beta cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient beta cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in beta cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic beta cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles.     

<Emphasis Type="Italic">Phellinus</Emphasis> extracts inhibit migration and matrix metalloproteinase secretion in porcine coronary artery endothelial cells

Phellinus linteus is a fungus which is found primarily in tropical regions of the Americas, Africa, and Asia.P. linteus has been used in traditional medical practice for the treatment of arthritis, liver damage and cancer. Angiogenesis is a process that involves migration, proliferation and cell differentiation, as well as the formation of new capillary structures. The anti-angiogenic activities evidenced by natural compounds may actually be a critical effect for the inhibition of angiogenesis-dependent disease by these agents via the blockage of vascular development. This study assessed the effects of water extracts fromP. linteus (Phellinus extracts) on primary cultured porcine coronary artery endothelial cells (PCAECs).Phellinus extracts induced no changes in DNA synthesis or cell numbers, but inhibited the migration of PCAECs.Phellinus extracts also induced a reduction in the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9. Our results show that, in endothelial cells,Phellinus extracts may inhibit angiogenesis by reducing levels of MMP-2 and MMP-9 secretion.