Detection of insulin-antibody binding on a solid surface using imaging ellipsometry

Imaging ellipsometry (IE) was used to detect the binding of insulin to its antibody on a solid surface. The modification of a gold surface with 11-mecaptoundecanoic acid (11-MUA), the adsorption of protein G, and antibody immobilization onto the protein G layer were confirmed by surface plasmon resonance. Ellipsometric images and ellipsometric angles of the surface antibody were acquired using the IE system by off-null ellipsometry. Ellipsometric images of antigen binding to the antibody were acquired, and their mean optical intensities estimated. Changes in mean optical intensity indicated that the detection range for insulin was from 10 ng/ml to 100 microg/ml.     

An integrated approach for thermal stabilization of a mesophilic adenylate kinase

Thermally stable proteins are desirable for research and industrial purposes, but redesigning proteins for higher thermal stability can be challenging. A number of different techniques have been used to improve the thermal stability of proteins, but the extents of stability enhancement were sometimes unpredictable and not significant. Here, we systematically tested the effects of multiple stabilization techniques including a bioinformatic method and structure‐guided mutagenesis on a single protein, thereby providing an integrated approach to protein thermal stabilization. Using a mesophilic adenylate kinase (AK) as a model, we identified stabilizing mutations based on various stabilization techniques, and generated a series of AK variants by introducing mutations both individually and collectively. The redesigned proteins displayed a range of increased thermal stabilities, the most stable of which was comparable to a naturally evolved thermophilic homologue with more than a 25° increase in its thermal denaturation midpoint. We also solved crystal structures of three representative variants including the most stable variant, to confirm the structural basis for their increased stabilities. These results provide a unique opportunity for systematically analyzing the effectiveness and additivity of various stabilization mechanisms, and they represent a useful approach for improving protein stability by integrating the reduction of local structural entropy and the optimization of global noncovalent interactions such as hydrophobic contact and ion pairs. Proteins 2014; 82:1947–1959. © 2014 Wiley Periodicals, Inc.     

Growth response of Pinus densiflora seedlings to different fertilizer compound ratios in a recently burned area in the eastern coast of Korea

This study was conducted to determine fertilizer compound ratios suitable for soil conditions and tree seedling growth in recently burned areas in Korean forests. Currently, the conventional fertilizer ratio applied to undisturbed forests in Korea is N:P:K 3:4:1. In this study, Japanese red pine (Pinus densiflora S. et Z.) seedlings planted in the burned area were fertilized over four growing seasons with the following NPK compound ratios: unfertilized (CON), 3:4:1, 6:4:1, 3:8:1, and 3:4:2. Fertilization generally increased current-year needle nutrient concentrations of the seedlings, and the chlorophyll a:b ratio in CON was significantly lower than in all fertilized plots. Fertilization significantly affected the growth of the pine seedlings, which had 71–87 % more height growth and 29–67 % increased root collar diameter (RCD) compared to CON. The increases in height and RCD were significantly higher with the 6:4:1 and 3:8:1 ratios than with the 3:4:1 ratio. The 3:4:2 and 3:4:1 fertilizer ratios had no effect on the RCD growth of seedlings. This suggests that the early growth of pine seedlings could be improved by providing high N and P supplies to areas affected by forest fires rather than the conventional fertilizer ratio of 3:4:1 in Korean forest soils. Therefore, application of the suitable fertilization ratio may be a very effective way to reduce reforestation cost as well as to shorten restoration period.     

Sequence polymorphisms in ribosomal RNA genes and variations in chromosomal loci of Oenothera odorata and O. laciniata

Numerous studies on Oenothera species have been investigated for the physiological and ecological characteristics. However, no such an information based on molecular cytogenetic has yet been introduced, in turn, is very essential for identifying sequence polymorphisms of rRNA genes with their loci on mitotic phases for further biological researches. In this study, sequence variations of rRNA genes in Oenothera odorata and O. laciniata were examined to identify informative factors as unique or repeat sequences in intra- and inter-specific variations. Intra-specific variation revealed that the sequences of the rRNA genes including the spacer regions were highly conserved revealing only a few variations. From the inter-specific variation, spacer regions of species were completely different as (1) non-homologous sequences in NTS and (2) different type repeat sequences in ITS 1, 2 and 5.8S rRNA, whereas the remaining coding regions were highly conserved. FISH was carried out on mitotic phases using the 5S rDNA of the analyzed sequences. From the interphase and metaphase chromosomes of the examined species, two loci of 5S rDNA in O. odorata and four loci in O. laciniata were confirmed on the telomeric region of the short arm. Due to the small size and unclear centromere of the chromosomes, karyotype could not be completed. However, we confirmed that the chromosomes are organized by meta- and acrocentric chromosomes and the chromosomes with identified loci were assumed to be paired by the location of loci at the telomeric region on the short arm with relative lengths.     

Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in confined compression.

Compression tests have often been performed to assess the biomechanical properties of full-thickness articular cartilage. We tested whether the apparent homogeneous strain-dependent properties, deduced from such tests, reflect both strain- and depth-dependent material properties. Full-thickness bovine articular cartilage was tested by oscillatory confined compression superimposed on a static offset up to 45%. and the data fit to estimate modulus, permeability, and electrokinetic coefficient assuming homogeneity. Additional tests on partial-thickness cartilage were then performed to assess depth- and strain-dependent properties in an inhomogeneous model, assuming three discrete layers (i = 1 starting from the articular surface, to i = 3 up to the subchondral bone). Estimates of the zero-strain equilibrium confined compression modulus (H(A0)), the zero-strain permeability (kp0) and deformation dependence constant (M), and the deformation-dependent electrokinetic coefficient (ke) differed among individual layers of cartilage and full-thickness cartilage. HiA0 increased from layer 1 to 3 (0.27 to 0.71 MPa), and bracketed the apparent homogeneous value (0.47 MPa). ki(p0) decreased from layer 1 to 3 (4.6 x 10(-15) to 0.50 x 10(-15) m2/Pa s) and was less than the homogeneous value (7.3 x 10(-15) m2/Pa s), while Mi increased from layer 1 to 3 (5.5 to 7.4) and became similar to the homogeneous value (8.4). The amplitude of ki(e) increased markedly with compressive strain, as did the homogeneous value: at low strain, it was lowest near the articular surface and increased to a peak in the middle-deep region. These results help to interpret the biomechanical assessment of full-thickness articular cartilage.     

Modulation of TNFSF expression in lymphoid tissue inducer cells by dendritic cells activated with Toll-like receptor ligands

Toll-like receptors (TLRs), which recognize structurally conserved components among pathogens, are mainly expressed by antigen-presenting cells such as dendritic cells (DCs), B cells, and macrophages. Recognition through TLRs triggers innate immune responses and influences antigen-specific adaptive immune responses. Although studies on the expression and functions of TLRs in antigen-presenting cells have been extensively reported, studies in lymphoid tissue inducer (LTi) cells have been limited. In this study, we observed that LTi cells expressed TLR2 and TLR4 mRNA as well as TLR2 protein and upregulated OX40L, CD30L, and TRANCE expression after stimulation with the TLR2 ligand zymosan or TLR4 ligand LPS. The expression of tumor necrosis factor superfamily (TNFSF) members was significantly upregulated when cells were cocultured with DCs, suggesting that upregulated TNFSF expression may contribute to antigen-specific adaptive immune responses.     

The Role in Ozone Phytotoxicity of the Evolution of Ethylene upon Induction of 1-Aminocydopropane-1-carboxylic Acid Synthase by Ozone Fumigation in Tomato Plants

The rate of evolution of ethylene by tomato plants was rapidlyincreased by O₃ fumigation. The time course of the increasein 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activitywas the same as that in the rate of evolution of ethylene, suggestingthat ACC synthase activity might be a rate-limiting step inthe evolution of ethylene that is caused by O₃ fumigation. Therate of the O₃-induced evolution of ethylene was increased bythe application of ACC to tomato plants, suggesting the involvementof ACC oxidase in the O₃-induced evolution of ethylene. Treatmentof plants with tiron inhibited the evolution of ethane, butnot of ethylene. These results indicated that evolution of ethylenein O₃-treated tomato plants might result from enzymatic reactionscatalyzed by both ACC synthase and ACC oxidase, but not fromstimulation by O₃ of the peroxidation of lipids mediated byfree radicals. Pretreatment of leaves with aminoethoxyvinylglycine (AVG), aninhibitor of ACC synthase, significantly inhibited the evolutionof ethylene that was induced by O₃ and concomitantly reducedthe extent of O₃-induced visible damage to leaves. Treatmentwith 2,5-norbonadiene, an inhibitor of the action of ethylene,strongly reduced the extent of visible damage caused by O₃,even though it did not suppress the evloution of ethylene. Theseresults indicate that ethylene acts on certain metabolic processesto cause visible damage. (Received September 7, 1995; Accepted December 18, 1995)     

The CD99 signal enhances Fas-mediated apoptosis in the human leukemic cell line, Jurkat

The CD99 antigen has been implicated in various cellular processes, including apoptosis in T cells. Previously, we reported two monoclonal antibodies that recognize different epitopes of the CD99 molecule, named DN16 and YG32. In this study, we investigated the role of each CD99 epitope in T cell apoptosis. Unlike the DN16 epitope, CD99 ligation via the YG32 epitope failed to induce T cell death. Surprisingly, however, the YG32 signal enhanced Fas-mediated apoptosis in Jurkat T cells. Augmentation of Fas-mediated apoptosis by YG32 ligation was inhibited by treatment with either of the caspase inhibitors z-VAD-fmk or z-IETD-fmk, and YG32 ligation appeared to induce Fas oligomerization. These results suggest that each CD99 epitope plays a distinct role in T cell biology, especially in T cell apoptosis.     

Effects of heat-killed <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> against influenza viruses in mice

The potential use of dietary measures to treat influenza can be an important alternative for those who lack access to influenza vaccines or antiviral drugs. Lactobacillus plantarum (Lp) is one of many lactic acid bacteria that grow in ‘kimchi’, an essential part of Korean meal, and several strains of Lp reportedly show protective effects against influenza. Using heat-killed Lp (nF1) isolated from kimchi, which is known for its immunomodulatory effects, we investigated whether regular oral intake of nF1 could influence the outcome of influenza virus infection in a mouse model. In a lethal challenge with influenza A (H1N1 and H3N2 subtypes) and influenza B (Yamagata lineage) viruses, daily oral administration of nF1 delayed the mean number of days to death of the infected mice and resulted in increased survival rates compared with those of the non-treated mice. Consistent with these observations, nF1 treatment also significantly reduced viral replication in the lungs of the infected mice. Taken together, our results might suggest the remedial potential of heatkilled Lactobacillus probiotics against influenza.