Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

Simulation of sequential batch reactor (SBR) operation for simultaneous removal of nitrogen and phosphorus

Modeling of the operation of sequential batch reactor (SBR) was performed to find out optimum design parameters for simultaneous removal of nitrogen and phosphorus in a small-scale wastewater treatment plant. The models were set up with material balances on SBR operation and Monod kinetics. The model parameters were obtained to best fit the experimental results in a small scale SBR. The models were useful in optimizing hydraulic retention time (HRT) and successfully simulated operations of SBR in a larger scale. Especially the model predicted well the reactions occurring in the filling period as well as the effect of dilution, and evaluated the performance of SBR process under diverse operating conditions.     

Engineering flavonoid glycosyltransferases for enhanced catalytic efficiency and extended sugar-donor selectivity

Flavonoids are predominantly found as glycosides in plants. The glycosylation of flavonoids is mediated by uridine diphosphate-dependent glycosyltransferases (UGT). UGTs attach various sugars, including arabinose, glucose, galactose, xylose, and glucuronic acid, to flavonoid aglycones. Two UGTs isolated from Arabidopsis thaliana, AtUGT78D2 and AtUGT78D3, showed 89 % amino acid sequence similarity (75 % amino acid sequence identity) and both attached a sugar to the 3-hydroxyl group of flavonols using a UDP-sugar. The two enzymes used UDP-glucose and UDP-arabinose, respectively, and AtUGT78D2 was approximately 90-fold more efficient than AtUGT78D3 when judged by the k _cat/K _m value. Domain exchanges between AtUGT78D2 and AtUGT78D3 were carried out to find UGTs with better catalytic efficiency for UDP-arabinose and exhibiting dual sugar selectivity. Among 19 fusion proteins examined, three showed dual sugar selectivity, and one fusion protein had better catalytic efficiency for UDP-arabinose compared with AtUGT78D3. Using molecular modeling, the changes in enzymatic properties in the chimeric proteins were elucidated. To the best of our knowledge, this is the first report on the construction of fusion proteins with expanded sugar-donor range and enhanced catalytic efficiencies for sugar donors.     

Comparison of multiple molecular dynamics trajectories calculated for the drug-resistant HIV-1 integrase T66I/M154I catalytic domain

HIV-1 integrase (IN) is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multidrug therapy of AIDS. Single and multiple mutations of IN at residues T66, S153, or M154 confer degrees of resistance to several inhibitors that prevent the enzyme from performing its normal strand transfer activity. Four different conformations of IN were chosen from a prior molecular dynamics (MD) simulation on the modeled IN T66I/M154I catalytic core domain as starting points for additional MD studies. The aim of this article is to understand the dynamic features that may play roles in the catalytic activity of the double mutant enzyme in the absence of any inhibitor. Moreover, we want to verify the influence of using different starting points on the MD trajectories and associated dynamical properties. By comparison of the trajectories obtained from these MD simulations we have demonstrated that the starting point does not affect the conformational space explored by this protein and that the time of the simulation is long enough to achieve convergence for this system.     

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.     

Neurogenesis of bone marrow-derived mesenchymal stem cells onto β-mercaptoethanol-loaded PLGA film

Bone marrow-derived mesenchymal stem cells (BMSCs) are of particular interest in the field of tissue engineering because of their potential to differentiate into osteoblasts, chondrocytes, and neuronal cells. In order to promote the differentiation of BMSCs into specific cell types, appropriate scaffold biomaterials and bioactive molecules that can support the differentiation of BMSCs into specific cell types are needed. We hypothesized that β-mercaptoethanol (BME), which has been reported to induce the differentiation of BMSCs into neural-like cells, promotes BMSCs to differentiate into neural-like cells when BME is added to polymeric scaffolds containing the BMSCs. We fabricated biocompatible film shaped scaffolds composed of poly(lacti-co-glycolic) acid (PLGA) and various concentrations of BME to confirm that BME-promoted differentiation of BMSCs is concentration-dependent. Cell proliferation increased as the BME concentration in the films increased at the early stage, and the proliferation rate remained similar on the PLGA films for 3 weeks following the BMSC seeding. The expression of neuronal markers in differentiated BMSCs was assessed by RT-PCR. At 2- and 3-week time-points, mRNA expression of neurofilament and neuron specific enolase was significantly increased in PLGA/BME films containing 400 μM BME compared to PLGA films. Thus, we have identified BMSC-seeded PLGA/BME films with 200 μM and 400 μM BME as potentially useful candidates for neural tissue engineering applications by promoting BMSC proliferation and differentiation towards neural-like cells.     

Synchronous Elicitation of Development in Root Caps Induces Transient Gene Expression Changes Common to Legume and Gymnosperm Species

Root cap development in cereals and legumes is self-regulated by a repressor that accumulates in the extracellular environment, and immersing the root tip into water results in renewed cap development. By exploiting this phenomenon, root cap mitosis and differentiation can be synchronously induced among populations. In Pisum sativum L., messenger RNA (mRNA) differential display revealed changes in expression of approximately 1% of the sample mRNA population within minutes of induced cap turnover. This profile changes sequentially over a period of 30 min, then stabilizes. Microarray analysis of Medicago truncatula root caps confirmed changes in expression of approximately 1% of the target population, within minutes. A cell specific marker for cap turnover exhibited the same temporal and spatial expression profile in the gymnosperm species Norway spruce (Picea abies) as in pea. Induced cap development provides a means to profile cell-specific gene expression among phylogenetically diverse species from the early moments of mitosis and cellular differentiation.     

QTL analysis of leaf morphology in tetraploid Gossypium (cotton)

Molecular markers were used to map and characterize quantitative trait loci (QTLs) determining cotton leaf morphology and other traits, in 180 F₂ plants from an interspecific cross between a Gossypium hirsutum genotype carrying four morphological mutants, and a wild-type Gossypium barbadense. The prominent effects of a single region of chromosome 15, presumably the classical ”Okra-leaf” locus, were modified by QTLs on several other chromosomes affecting leaf size and shape. For most traits, each parent contained some alleles with positive effects and others with negative effects, suggesting a large potential for adapting leaf size and shape to the needs of particular production regimes. Twenty one QTLs/loci were found for the morphological traits at LOD≥3.0 and P≤0.001, among which 14 (63.6%) mapped to D-subgenome chromosomes. Forty one more possible QTLs/loci were suggested with 2.0≤LOD<3.0 and 0.001<P≤0.01. Among all of the 62 possible QTLs (found at LOD≥2.0 and P≤0.01) for the 14 morphological traits in this study, 38 (61.3%) mapped to D-subgenome chromosomes. This reinforces the findings of several other studies in suggesting that the D-subgenome of tetraploid cotton has been subject to a relatively greater rate of evolution than the A-subgenome, subsequent to polyploid formation. Received: 26 April 1999 / Accepted: 30 July 1999