Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

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.     

Identification and characterization of acetyl-CoA carboxylase gene cluster in <Emphasis Type="Italic">Streptomyces toxytricini</Emphasis>

The gene locus for acetyl-CoA carboxylase (ACC) involved in the primary metabolism was identified from the genomic library of Streptomyces toxytricini which produces a lipase inhibitor lipstatin. The 7.4 kb cloned gene was comprised of 5 ORFs including accD1, accA1, hmgL, fadST1, and stsF. In order to confirm the biochemical characteristics of AccA1, the gene was overexpressed in Escherichia coli cells, and the recombinant protein was purified through Ni²⁺ affinity chromatography. Because most of the expressed AccAl was biotinylated by host E. coli BirA in the presence of D-biotin, the non-biotinylated apo-AccA1 was purified after gene induction without D-biotin, followed by exclusion of holo-AccA1 using streptavidin beads. The separated apo-AccA1 was post-translationally biotinylated by S. toxytricini biotin apo-protein ligase (BPL) in a time- and enzyme-dependent manner. This result supports that this gene cluster of S. toxytricini encodes the functional ACC enzyme subunits to be biotinylated.     

Genetic and Molecular Studies for Regulation of Bolting Time of Onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.)

The control of bolting time in onion is an important approach for bulb and seed production, as onion plants which bolt do not produce marketable bulbs and seed yields are dependent on floral induction. However, genetic and molecular studies about bolting time in onion plants have not been examined yet to date. In order to understand the regulation of bolting time in onion plants, we conducted the genetic crosses between late bolting-type cultivar (MOS8) and very early bolting-type cultivar (Guikum). Segregation ratio of late to very early in F₂ populations indicated that this lateness trait was determined by a dominant locus. We also analyzed protein profiles in onion plants with different bolting time by a proteomics approach. Interestingly, a protein spot with significant similarities to chromodomains of mammalian chromo-ATPase/helicase-DNA-binding 1 or heterochromatin protein 1, which is involved in the histone modifications, was identified. Histone methyltransferase activity was also observed in onion plants. Taken together, these results suggest that a genetic pathway may be involved in the modulation of bolting time in onion plants, though there is no direct evidence that this protein spot obtained by proteomics is relevant to vernalization.     

Anaerobic ammonia-oxidation coupled with Fe3+ reduction by an anaerobic culture from a piggery wastewater acclimated to NH4 +/Fe3+ medium

The oxidation of ammonia coupled with the reduction of iron is a unique pathway mostly reported in soils and sediments. An anaerobic sludge from a piggery wastewater treatment plant had been acclimated to an NH⁺/Fe³⁺-rich environment to secure an enrichment culture and investigate an anaerobic ammonia oxidation coupled with an iron reduction. The enrichment culture showed an average pH of 6.8 and the concentration of mixed liquor volatile suspended solid was measured as 1,120 mg/L. The mol ratio of oxidized NH₄ ⁺ and reduced Fe³⁺ was 0.33 mol NH₄ ⁺/mol Fe³⁺. It was suggested that the culture acclimated to NH₄ ⁺/Fe³⁺ contained the anaerobic ammonia oxidizing bacteria as well and thus NH₄ ⁺ was fully oxidized to NO₃ ⁻ by the bacterial consortia. In a batch experiment using the culture, the oxidation of NH₄ ⁺ was increased as the initial concentration increased. However, it was suspected from the experimental results that other iron reducing bacteria had grown under the environment applied for the enrichment culture. As a result, it was observed that heterotrophic and autotrophic iron reducers were competing for Fe³⁺.     

The growth-inhibitory effects of pawpaw (Asimina triloba [L.] Dunal) roots,twigs, leaves,and fruit against human gastric (AGS) and cervical (HeLa) cancer cells and their anti-inflammatory activities

Molecular Biology Reports - The pawpaw tree has several beneficial effects. However, no studies have been conducted to address the mechanisms underlying the cytotoxic effects of pawpaw extracts...     

Hair Tissue Mineral Analysis and Metabolic Syndrome

Deficiency of minerals causes functional abnormality of enzymes, frequently resulting in metabolic disturbance. We investigated possible relationship between minerals and metabolic syndrome by analysis of hair tissue minerals. We selected 848 subjects older than 20 years of age at Ajou University Hospital from May 2004 to February 2007. We excluded the subjects who had cancers, steroid and thyroid medication, and incomplete record from the study. Finally, 343 subjects were eligible. We performed cross-sectional analysis for the relationship between minerals and metabolic syndrome. The contents of calcium, magnesium, and copper in the metabolic syndrome group were significantly lower than those of the normal group, whereas the amounts of sodium, potassium, and mercury in the metabolic syndrome group were significantly higher than those of the normal group. By dividing the subjects into quartile with the level of calcium, magnesium, and mercury concentrations, we carried out logistic regression analysis to study the subjects and found that the subjects in the third quartile of calcium and magnesium concentrations had significantly lower odds ratio (OR) of the metabolic syndrome compared with that of the lowest quartile group [OR = 0.30, confidence interval (CI) = 0.10–0.89; OR = 0.189, CI = 0.063–0.566] and that the subjects in the highest mercury quartile had significantly higher OR of the metabolic syndrome compared with that of the lowest mercury quartile group (OR = 7.35, CI = 1.73–31.1). As part of the metabolic syndrome, the optimal calcium and magnesium concentrations in hair tissue may reflect decreased risk of metabolic syndrome, whereas high mercury concentration in hair tissue may indicate increased risk of metabolic syndrome.     

A flow injection analysis system with encapsulated high-density <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells for rapid determination of biochemical oxygen demand

The biochemical oxygen demand (BOD) determination was studied using a novel flow injection analysis (FIA) system with encapsulated Saccharomyces cerevisiae cells and an oxygen electrode and was compared with conventional 5-day BOD tests. S. cerevisiae cells were packed in a calcium alginate capsule at a dry cell weight of 250 g/l of capsule core. The level of dissolved oxygen (DO) was reduced due to the enhanced respiratory activity of the microbial cells when the injected nutrient passed through the bioreactor. The decrease in DO (ΔDO) was intensified with the amount of microbial cells packed in the bioreactor. However, the specific ΔDO decreased as the amount of cells loaded in the bioreactor increased. The ΔDO value was dependent on the pH and temperature of the mobile phase and reached its maximum value at 35°C and pH 7–8. Also, ΔDO became larger at longer response times as the flow rate of the mobile phase decreased. The measurement of ΔDO was repeated more than six times consecutively using a 20-ppm standard glucose and glutamic acid solution, which confirmed the reproducibility with a standard deviation of 0.95%. A strong linear correlation between ΔDO and BOD was also observed. The 5-day BOD values of actual water and wastewater samples were in accordance with the BOD values obtained by this FIA method using encapsulated S. cerevisiae cells. Unlike the cell-immobilized bead system, there was no contamination of the bioreactor resulting from any leak of yeast cells from the sensor capsules during BOD measurements.     

Neurite Outgrowth Effect of 4-O-methylhonokiol by Induction of Neurotrophic Factors Through ERK Activation

Compounds isolated from Magnolia officinalis such as magnolol, honokiol and obovatol exhibit several pharmacological effects on CNS including depressant, anxiolytic and anticonvulsant effects, as well as neuroprotective effects against chemical and heat damages. Recently, honokiol was found to have a neurotrophic effect in fetal rat cortical neurons. In the present study, we show that 4-O-methylhonokiol, a novel compound from Magnolia officinalis, promotes neurite outgrowth in a concentration-dependent manner in rat embryonic neuronal cells. In parallel with the neurite outgrowth activity, the expression of neurite outgrowth marker proteins is also increased by treatment with 4-O-methylhonokiol. We also found that 4-O-methylhonokiol promotes the release of NGF and BDNF into cell culture medium. In addition, lower concentration of 4-O-methylhonokiol (1 and 2 μM) further enhanced neurite outgrowth and expression of neurite outgrowth marker proteins in the presence of NGF (50 ng/ml) or BDNF (10 ng/ml). Subsequently, we found that 4-O-methylhonokiol activates ERK in a concentration-dependent manner. However, the neurite outgrowth activity and the NGF and BDNF release induced by 4-O-methylhonokiol are suppressed by an ERK-specific inhibitor. These results suggest that 4-O-methylhonokiol has the ability to induce neurite outgrowth via the increase of neurotrophic factor levels through ERK activation.