Enzymatic synthesis of 5-bromo-2’-deoxyuridine-2-14C and of 5-iodo-2’-deoxyuridine-2-14C and their incorporation into deoxyribonucleic acid (Allium cepa)

5-Bromo-2’-deoxyuridine-2-¹⁴C was prepared from 5-bromouracil-2-¹⁴C and 2’-de-oxyguanosine using trans-N-deoxyribosylase fromLactobacillus helveticus and incorporated into DNA ofAllium cepa roots. After isolating the DNA and hydrolyzing it enzymatically to deoxynucleoside-5’-phosphates a radioactive nucleotide was detected which yielded 5-bromo-2’-deoxyuridine-2-¹⁴C on enzymatic dephosphorylation. The incorporation of 5-iodo-2’-deoxy-uridine-2-¹⁴C was followed only by microautoradiography.     

Lipopolysaccharide and interferon-gamma-induced nitric oxide production and protein oxidation in mouse peritoneal macrophages are affected by glutathione peroxidase-1 gene knockout

This study investigated the role of glutathione peroxidase-1 (GPX1) in protein oxidation in peritoneal macrophages. Macrophages isolated from both wild-type (WT) and GPX1 knockout (KO) mice were activated by lipopolysaccharide (LPS, 1 microg/ml) and interferon-gamma (IFN, 10 U/ml for 24 or 48 h in the presence or absence of 1 microM diquat (DQ), 250 microM aminoguanidine (AG, an inhibitor of inducible nitric oxide synthase), and (or) 100 microM diethyldithiocarbamate (DETC, an inhibitor of Cu,Zn-SOD). In the KO macrophages, there was no protein band detected by Western blot with anti-GPX1 antibody and 98% reduction in total GPX activity compared with WT cells. Nitric oxide (NO) synthesis was greatly enhanced after 24 h by GPX1 knockout and DQ, but inhibited by AG or DETC. Protein carbonyl formation in total cell extract was clearly associated with NO synthesis as higher levels of protein carbonyl were detected in activated KO than WT macrophages, and DQ enhanced slightly while AG or DETC virtually blocked its formation. A similarly marginal effect of GPX1 KO was observed on protein nitration. The LPS/IFN/DQ-induced DNA fragmentation was blocked by AG, but not by DETC. Cell viability at 48 h was decreased by the LPS/IFN activation and further reduced by the addition of DQ, but restored by AG. In conclusion, GPX1 affects the NO production in activated peritoneal macrophages and protects these cells against NO-associated protein oxidation.     

A Dual Tracer 18F-FCH/18F-FDG PET Imaging of an Orthotopic Brain Tumor Xenograft Model

Early diagnosis of low grade glioma has been a challenge to clinicians. Positron Emission Tomography (PET) using ¹⁸F-FDG as a radio-tracer has limited utility in this area because of the high background in normal brain tissue. Other radiotracers such as ¹⁸F-Fluorocholine (¹⁸F-FCH) could provide better contrast between tumor and normal brain tissue but with high incidence of false positives. In this study, the potential application of a dual tracer ¹⁸F-FCH/¹⁸F-FDG-PET is investigated in order to improve the sensitivity of PET imaging for low grade glioma diagnosis based on a mouse orthotopic xenograft model. BALB/c nude mice with and without orthotopic glioma xenografts from U87 MG-luc2 glioma cell line are used for the study. The animals are subjected to ¹⁸F-FCH and ¹⁸F-FDG PET imaging, and images acquired from two separate scans are superimposed for analysis. The ¹⁸F-FCH counts are subtracted from the merged images to identify the tumor. Micro-CT, bioluminescence imaging (BLI), histology and measurement of the tumor diameter are also conducted for comparison. Results show that there is a significant contrast in ¹⁸F-FCH uptake between tumor and normal brain tissue (2.65 ± 0.98), but with a high false positive rate of 28.6%. The difficulty of identifying the tumor by ¹⁸F-FDG only is also proved in this study. All the tumors can be detected based on the dual tracer technique of ¹⁸F-FCH/ ¹⁸F-FDG-PET imaging in this study, while the false-positive caused by ¹⁸F-FCH can be eliminated. Dual tracer ¹⁸F-FCH/¹⁸F-FDG PET imaging has the potential to improve the visualization of low grade glioma. ¹⁸F-FCH delineates tumor areas and the tumor can be identified by subtracting the ¹⁸F-FCH counts. The sensitivity was over 95%. Further studies are required to evaluate the possibility of applying this technique in clinical trials.     

Does Marriage Make Us Healthier? Inter-Country Comparative Evidence from China,Japan, and Korea

Objectives

This study focuses on East Asian countries and investigates the difference in the marriage premium on the health-marriage protection effect (MPE) between younger and older generations and the intra-couple education concordance effect (ECE) on the health of married individuals. This study used inter-country comparative data from China, Japan, and Korea.

Methods

This study focused on individuals (n = 7,938) in China, Japan, and Korea who were sampled from the 2010 East Asian Social Survey. To investigate MPE and ECE, four health indicators were utilized: a physical and mental components summary (PCS and MCS), self-rated health status (D_self), and happiness level (D_happy). Ordinary least squares regression was conducted by country- and gender-specific subsamples.

Results

We found that the MPE on PCS, MCS, and D_self was more significant for the older generation than for the younger generation in both China and Japan, whereas the results were inconclusive in Korea. With regard to the ECE on happiness (D_happy), for both men and women, couples tend to be happier when both the husband and the wife are well educated (“higher balanced marriage”) compared to couples with a lower level of educational achievement (“lower balanced marriage”). Significant benefits from a “higher balanced marriage” on MCS and D_self were observed for women only. In contrast, no statistically significant differences in health status were observed between “higher balanced marriage” couples and couples with different levels of educational achievements (“upward marriage” or “downward marriage”).

Conclusions

This study found that (1) the MPE was more significant for the older generation, and (2) the health gap, particularly the happiness gap, between higher- and lower-balanced married couples was significant. The inter-country comparative findings are useful to explain how the role of marriage (and therefore of family) on health has been diluted due to the progress of industrialization and modernization.     

利用激光微束穿刺法将外源基因导入小麦的研究

用激光微束穿刺法将携带有新霉素磷酸转移酶（ＮＰＴ－Ⅱ）基因的质粒ｐＪＩＴ１０１导入京花１号小麦幼胚细胞。方法是将小麦幼胚细胞进行高渗缓冲液预处理,用微米级的激光微束处理,然后在卡那霉素培养基上筛选出具抗性的愈伤组织及绿色小植株。第一年,从１５０个小麦幼胚中,在卡那霉素培养基上筛选出４株绿苗,取两株进行ＮＰＴ－Ⅱ酶活性分析,测到了ＮＰＴ－Ⅱ酶的活性。第二年重复实验,从２４５个小麦幼胚中,经筛选获得１株绿苗,进行了叶片ＤＮＡＰＣＲ扩增检测,转化的绿色小苗扩增出所导入的ＮＰＴ－Ⅱ基因编码的片段。结果表明,外源ＮＰＴ－Ⅱ基因已导入了小麦,并实现了整合表达。     

Noninvasive Evaluation of Metabolic Tumor Volume in Lewis Lung Carcinoma Tumor-Bearing C57BL/6 Mice with Micro-PET and the Radiotracers 18F-Alfatide and 18F-FDG: A Comparative Analysis

Purpose

To explore the value of a new simple lyophilized kit for labeling PRGD₂ peptide (¹⁸F-ALF-NOTA-PRGD₂, denoted as ¹⁸F-alfatide) in the determination of metabolic tumor volume (MTV) with micro-PET in lewis lung carcinoma (LLC) tumor-bearing C57BL/6 mice verified by pathologic examination and compared with those using ¹⁸F-fluorodeoxyglucose (FDG) PET.

Methods

All LLC tumor-bearing C57BL/6 mice underwent two attenuation-corrected whole-body micro-PET scans with the radiotracers ¹⁸F-alfatide and ¹⁸F-FDG within two days. ¹⁸F-alfatide metabolic tumor volume (V_RGD) and ¹⁸F-FDG metabolic tumor volume (V_FDG) were manually delineated slice by slice on PET images. Pathologic tumor volume (V_Path) was measured in vitro after the xenografts were removed.

Results

A total of 37 mice with NSCLC xenografts were enrolled and 33 of them underwent ¹⁸F-alfatide PET, and 35 of them underwent ¹⁸F-FDG PET and all underwent pathological examination. The mean ± standard deviation of V_Path, V_RGD, and V_FDG were 0.59±0.32 cm³ (range,0.13~1.64 cm³), 0.61±0.37 cm³ (range,0.15~1.86 cm³), and 1.24±0.53 cm³ (range,0.17~2.20 cm³), respectively. V_Path vs. V_RGD, V_Path vs. V_FDG, and V_RGD vs. V_FDG comparisons were t = -0.145, P = 0.885, t = -6.239, P<0.001, and t = -5.661, P<0.001, respectively. No significant difference was found between V_Path and V_RGD. V_FDG was much larger than V_RGD and V_Path. V_RGD seemed more approximate to the pathologic gross tumor volume. Furthermore, V_Path was more strongly correlated with V_RGD (R = 0.964,P<0.001) than with V_FDG (R = 0.584,P<0.001).

Conclusions

¹⁸F-alfatide PET provided a better estimation of gross tumor volume than ¹⁸F-FDG PET in LLC tumor-bearing C57BL/6 mice.     

ZAKβ antagonizes and ameliorates the cardiac hypertrophic and apoptotic effects induced by ZAKα

ZAK (sterile alpha motif and leucine zipper containing kinase AZK), a serine/threonine kinase with multiple biochemical functions, has been associated with various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy. In our previous reports, we found that the activation of ZAKα signaling was critical for cardiac hypertrophy. In this study, we show that the expression of ZAKα activated apoptosis through both a FAS‐dependent pathway and a mitochondria‐dependent pathway by subsequently inducing caspase‐3. ZAKβ, an isoform of ZAKα, is dramatically expressed during cardiac hypertrophy and apoptosis. The interaction between ZAKα and ZAKβ was demonstrated here using immunoprecipitation. The results show that ZAKβ has the ability to diminish the expression level of ZAKα. These findings reveal an inherent regulatory role of ZAKβ to antagonize ZAKα and to subsequently downregulate the cardiac hypertrophy and apoptosis induced by ZAKα.     

Flow‐injection chemiluminescence method to detect a β2 adrenergic agonist

A new method for the detection of β₂ adrenergic agonists was developed based on the chemiluminescence (CL) reaction of β₂ adrenergic agonist with potassium ferricyanide–luminol CL. The effect of β₂ adrenergic agonists including isoprenaline hydrochloride, salbutamol sulfate, terbutaline sulfate and ractopamine on the CL intensity of potassium ferricyanide–luminol was discovered. Detection of the β₂ adrenergic agonist was carried out in a flow system. Using uniform design experimentation, the influence factors of CL were optimized. The optimal experimental conditions were 1 mmol/L of potassium ferricyanide, 10 µmol/L of luminol, 1.2 mmol/L of sodium hydroxide, a flow speed of 2.6 mL/min and a distance of 1.2 cm from ‘Y₂’ to the flow cell. The linear ranges and limit of detection were 10–100 and 5 ng/mL for isoprenaline hydrochloride, 20–100 and 5 ng/mL for salbutamol sulfate, 8–200 and 1 ng/mL for terbutaline sulfate, 20–100 and 4 ng/mL for ractopamine, respectively. The proposed method allowed 200 injections/h with excellent repeatability and precision. It was successfully applied to the determination of three β₂ adrenergic agonists in commercial pharmaceutical formulations with recoveries in the range of 96.8–98.5%. The possible CL reaction mechanism of potassium ferricyanide–luminol–β₂ adrenergic agonist was discussed from the UV/vis spectra. Copyright © 2014 John Wiley & Sons, Ltd.