Supplementing with L-Tryptophan Increases Medium Protein and Alters Expression of Genes and Proteins Involved in Milk Protein Synthesis and Energy Metabolism in Bovine Mammary Cells

The objective of this study was to investigate the effects of supplementing with L-tryptophan (L-Trp) on milk protein synthesis using an immortalized bovine mammary epithelial (MAC-T) cell line. Cells were treated with 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM of supplemental L-Trp, and the most efficient time for protein synthesis was determined by measuring cell, medium, and total protein at 0, 24, 48, 72, and 96 h. Time and dose tests showed that the 48 h incubation time and a 0.9 mM dose of L-Trp were the optimal values. The mechanism of milk protein synthesis was elucidated through proteomic analysis to identify the metabolic pathway involved. When L-Trp was supplemented, extracellular protein (medium protein) reached its peak at 48 h, whereas intracellular cell protein reached its peak at 96 h with all L-Trp doses. β-casein mRNA gene expression and genes related to milk protein synthesis, such as mammalian target of rapamycin (mTOR) and ribosomal protein 6 (RPS6) genes, were also stimulated (p < 0.05). Overall, there were 51 upregulated and 59 downregulated proteins, many of which are involved in protein synthesis. The results of protein pathway analysis showed that L-Trp stimulated glycolysis, the pentose phosphate pathway, and ATP synthesis, which are pathways involved in energy metabolism. Together, these results demonstrate that L-Trp supplementation, particularly at 0.9 mM, is an effective stimulus in β-casein synthesis by stimulating genes, proteins, and pathways related to protein and energy metabolism.     

Effects of Adenine Methylation on the Structure and Thermodynamic Stability of a DNA Minidumbbell

DNA methylation is a prevalent regulatory modification in prokaryotes and eukaryotes. N¹-methyladenine (m¹A) and N⁶-methyladenine (m⁶A) have been found to be capable of altering DNA structures via disturbing Watson–Crick base pairing. However, little has been known about their influences on non-B DNA structures, which are associated with genetic instabilities. In this work, we investigated the effects of m¹A and m⁶A on both the structure and thermodynamic stability of a newly reported DNA minidumbbell formed by two TTTA tetranucleotide repeats. As revealed by the results of nuclear magnetic resonance spectroscopic studies, both m¹A and m⁶A favored the formation of a T·m¹A and T·m⁶A Hoogsteen base pair, respectively. More intriguingly, the m¹A and m⁶A modifications brought about stabilization and destabilization effects on the DNA minidumbbell, respectively. This work provides new biophysical insights into the effects of adenine methylation on the structure and thermodynamic stability of DNA.     

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC₅₀ of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.     

Material Design for 3D Multifunctional Hydrogel Structure Preparation

Hydrogels are recognized as one of the most promising materials for e-skin devices because of their unique applicable functionalities such as flexibility, stretchability, biocompatibility, and conductivity. Beyond the excellent sensing functionalities, the e-skin devices further need to secure a target-oriented 3D structure to be applied onto various body parts having complex 3D shapes. However, most e-skin devices are still fabricated in simple 2D film-type devices, and it is an intriguing issue to fabricate complex 3D e-skin devices resembling target body parts via 3D printing. Here, a material design guideline is provided to prepare multifunctional hydrogels and their target-oriented 3D structures based on extrusion-based 3D printing. The material design parameters to realize target-oriented 3D structures via 3D printing are systematically derived from the correlation between material design of hydrogels and their gelation characteristics, rheological properties, and 3D printing processability for extrusion-based 3D printing. Based on the suggested material design window, ion conductive self-healable hydrogels are designed and successfully applied to extrusion-based 3D printing to realize various 3D shapes.     

Determination of non-methane hydrocarbon emission factors from vehicles in a Tunnel in Seoul in May 2000

Measurements of non-methane hydrocarbons (NMHC) were performed at the entrance and exit of the Sangdo tunnel to estimate emission factors (EF) of NMHC from vehicles in May 2000. About 50 species were analyzed by a combined GC/FID and GC/MS system. Ethylene was the most abundant compound, followed by n-butane and acetylene, respectively. Based on the measurement data, the real world vehicular EF in Seoul was estimated. The highest EF value was 89.8 mg (veh-mile)^-1 for n-butane, followed by ethylene and toluene.     

Catalytic Combustion of Methane over Rare Earth Stannate Pyrochlore

Well-defined Ln₂Sn₂O₇ powders (Ln = La, Sm and Gd) with a phase-pure pyrochlore structure were synthesized by hydrothermal reaction. The catalytic activities of Ln₂Sn₂O₇ powders for methane combustion were measured. Methane oxidation started at 500 °C and increased with oxidation temperature. Catalytic methane combustion is strongly influenced by the presence of oxygen vacancies that form by breaking Sn–O lattice bonds as the temperature increases. Addition of manganese to the rare earth pyrochlores improved methane oxidation activity. Manganese-doped samarium stannate pyrochlore (Sm₂Sn_1.8Mn_0.2O₇) shows highest the catalytic activity. Light-off and complete oxidation temperatures were measured at about 400 and 650 °C, respectively.     

An experimental investigation of mixing of carbon nanotube/polymer composite in a batch-type screw mixer

We develop a miniaturized batch-type screw mixer (BSM) for uniform mixing of polymer resin and nanoparticles, based on the stretching of material elements. This stretching is induced by the combination of recirculating cross-sectional flows in deep channels of the screw and high shear stress developed at flight regions. The BSM is used to produce a polymer nano-composite composed of multi-walled carbon nanotubes and polydimethylsiloxane resin. The mixing performance of the BSM is characterized quantitatively by estimating two different types of mixing efficiencies (i.e., dispersive mixing and distributive mixing) via transmitted light microscope images. The developed BSM highly improves the mixing performance rather than that of a conventional ultrasonic mixing device.     

Selective Oxidation of Methanol to Formaldehyde Using Modified Iron-Molybdate Catalysts

Methanol selective oxidation to formaldehyde over a modified Fe-Mo catalyst with two different stoichiometric (Mo/Fe atomic ratio = 1.5 and 3.0) was studied experimentally in a fixed bed reactor over a wide range of reaction conditions. The physicochemical characterization of the prepared catalysts provides evidence that Fe₂(MoO₄)₃ is in fact the active phase of the catalyst. The experimental results of conversion of methanol and selectivity towards formaldehyde for various residence times were studied. The results showed that as the residence time increases the yield of formaldehyde decreases. Selectivity of formaldehyde decreases with increase in residence time. This result is attributable to subsequent oxidation of formaldehyde to carbon monoxide due to longer residence time.     

Preparation and properties of polyelectrolyte complex sponges composed of hyaluronic acid and chitosan and their biological behaviors

Polyelectrolyte complexes (PECs) composed of chitosan and hyaluronic acid (HA) were prepared in various pH regions and at different weight ratios. At low pHs, there was a strong ionic interaction between NH groups in chitosan and both COO^? and COOH groups in HA due to the deprotonation of HA, whereas weak linkages were formed at high pHs because only the carboxyl groups of HA could interact with NH groups in chitosan. The formation of PECs resulted in a decrease in the crystallinity and thermal stability caused by the interactions between polyions. With variations in the degree of ionization of polyions at various pH conditions, novel PEC sponges were prepared by the freeze drying of PEC solutions. Furthermore, for the evaluation of the wound‐healing effect of PEC sponges with or without an antimicrobial agent (silver sulfadiazine), they were applied to a full‐skin defect of a Wistar rat in vivo. The histology and computerized morphometric analysis of the epidermal healing confirmed the proliferation of fibroblasts in the wound bed and a distinct reduction in infectious agents. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 925–932, 2003