Stable Agrobacterium-mediated transformation of the halophytic Leymus chinensis (Trin.)

In this study, an efficient procedure for stable Agrobacterium-mediated transformation of Leymus chinensis (Trin.) was established. Agrobacterium tumefaciens strain EHA105, harboring a binary vector pCAMBIA2300, was used for transformation, along with a sweet potato 2-cysteine peroxiredoxin (2-Cys Prx) gene under the control of the stress-inducible sweet potato anionic peroxidase 2 (SWPA2) promoter and the neomycin phosphotransferase (nptII) gene under the control of the cauliflower mosaic virus (CaMV) 35 S promoter. We found that a one-month-old callus derived from mature seeds could be efficiently transformed. Seven-day preculture followed by inoculation with the addition of 100 μmolL-1 acetosyringone (AS) and then a 3 day co-cultivation were performed before selection. Selection of transgenic shoots was done in the presence of 150 mgL-1 kanamycin (KM). An optical density at a wavelength of 600 nm (OD600) of approximately 0.4 for A. tumefaciens infection solution and 20 min of infection time gave the highest transformation efficiency. Polymerase chain reaction (PCR) analysis of KM-resistant plants and newly regenerated rhizomes revealed stable transformation of the 2-Cys Prx gene and the nptII gene, with the highest transformation frequency of 4.93%. RT-RCR analysis was conducted using salt stressed transgenic plants, and the results suggested that 2-Cys Prx had low transcription levels under non-stressed conditions, and increased transcription after 6 h of 200 mM NaCl stress. This gene continued to demonstrate high levels of transcription until 6 h after withdrawal of stress, with a slow recovery. The method reported herein provides a direct opportunity for improvement of the quality traits of L. chinensis via genetic transformation.Keywords: Leymus chinensis, Agrobacterium-mediated transformation, 2-Cys peroxiredoxin, gene transformatio

DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene

Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5–8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5–100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs

A Higher Burden of Small Low-density Lipoprotein Particles is Associated with Profound Changes in the Free Androgen Index in Male Adolescents

From a young age, males are at higher cardiovascular risk than females. Dyslipidemia, including a higher burden related to small low-density lipoproteins (LDL), plays an important role in precipitating atherosclerosis in both males and females. We investigated sex differences in atherogenic lipoprotein burden and the independent predictors of LDL particle size in children and adolescents. We measured the concentrations of total testosterone, sex hormone-binding globulin, estradiol, total cholesterol, triglyceride, LDL cholesterol, HDL cholesterol, and LDL particle size in 135 children and adolescents (67 boys, 68 girls). The free androgen index was significantly and negatively correlated with LDL particle size (r = -0.273, P = 0.026) in boys, but estrogen and LDL particle size were not related. In a stepwise multiple regression analysis adjusted for body mass index, age, and homeostasis model assessment for insulin resistance, free androgen index was still an independent predictor of LDL particle size in boys (R2 = 0.075, P = 0.026). The prominent decrease in LDL particle size along with increased testosterone concentrations in males might explain why they are more likely to display atherogenic dyslipidemia from adolescence

Inflammatory and Remodeling Events in Asthma with Chronic Exposure to House Dust Mites: A Murine Model

Although animal models with ovalbumin have been used to study chronic asthma, there are difficulties in inducing recurrence as well as in maintaining chronic inflammation in this system. Using a murine model of house dust mite (HDM)-induced bronchial asthma, we examined the airway remodeling process in response to the chronic exposure to HDM. During the seventh and twelfth weeks of study, HDM were inhaled through the nose for three consecutive days and airway responsiveness was measured. Twenty-four hours later, bronchoalveolar lavage and histological examination were performed. The degree of overproduction of mucus, subepithelial fibrosis, and the thickness of the peribronchial smooth muscle in the experimental group was clearly increased compared to the control group. In addition, HDM-exposed mice demonstrated severe airway hyperreactivity to methacholine. In the bronchoalveolar lavage fluid, the number of total cells and eosinophils was increased; during the twelfth week, the number of neutrophils increased in the experimental group. With regard to changes in cytokines, the concentrations of IL-4, IL-13, and transforming growth factor-beta (TGF-β) were increased in the experimental group. The data suggest that eosinophils, IL-4, IL-13, and TGF-β might play an important role in the airway remodeling process and that neutrophils may be involved with increased exposure time

Time Sequence of Airway Remodeling in a Mouse Model of Chronic Asthma: the Relation with Airway Hyperresponsiveness

During the course of establishing an animal model of chronic asthma, we tried to elucidate the time sequence of airway hyperresponsiveness (AHR), airway inflammation, airway remodeling, and associated cytokines. Seven-week-old female BALB/c mice were studied as a chronic asthma model using ovalbumin (OVA). After sensitization, mice were exposed twice weekly to aerosolized OVA, and were divided into three groups depending on the duration of 4 weeks, 8 weeks, and 12 weeks. At each time point, airway responsiveness, inflammatory cells, cytokines in bronchoalveolar lavage fluids (BALF), serum OVA-specific IgE, IgG1, IgG2a, and histological examination were carried out. AHR to methacholine, increased levels of OVA-specific IgG1 and IgG2a, and goblet cell hyperplasia were continuously sustained at each time point of weeks. In contrast, we observed a time-dependent decrease in serum OVA-specific IgE, BALF eosinophils, BALF cytokines such as IL-13, transforming growth factor-beta1, and a time-dependent increase in BALF promatrix metalloproteinase-9 and peribronchial fibrosis. In this OVA-induced chronic asthma model, we observed airway remodelings as well as various cytokines and inflammatory cells being involved in different time-dependent manners. However, increased airway fibrosis did not directly correlate with a further increase in airway hyperresponsiveness