Nitroalkenes confer acute cardioprotection via adenine nucleotide translocase 1

Electrophilic nitrated lipids (nitroalkenes) are emerging as an important class of protective cardiovascular signaling molecules. Although species such as nitro-linoleate (LNO(2)) and nitro-oleate can confer acute protection against cardiac ischemic injury, their mechanism of action is unclear. Mild uncoupling of mitochondria is known to be cardioprotective, and adenine nucleotide translocase 1 (ANT1) is a key mediator of mitochondrial uncoupling. ANT1 also contains redox-sensitive cysteines that may be targets for modification by nitroalkenes. Therefore, in this study we tested the hypothesis that nitroalkenes directly modify ANT1 and that nitroalkene-mediated cardioprotection requires ANT1. Using biotin-tagged LNO(2) infused into intact perfused hearts, we obtained mass spectrometric (MALDI-TOF-TOF) evidence for direct modification (nitroalkylation) of ANT1 on cysteine 57. Furthermore, in a cell model of ischemia-reperfusion injury, siRNA knockdown of ANT1 inhibited the cardioprotective effect of LNO(2). Although the molecular mechanism linking ANT1-Cys(57) nitroalkylation and uncoupling is not yet known, these data suggest that ANT1-mediated uncoupling may be a mechanism for nitroalkene-induced cardioprotection.     

Identification,expression, and characterization of the highly conserved D-xylose isomerase in animals

D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway？ With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase （EC 5.3.1.5）. The xyiose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg^2＋. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.     

Identification of hemorphins in a cathepsin D bovine hemoglobin hydrolysate by radioimmunoassay and photodiode array detections

Summary Morphinomimetic peptides have been purified from hemoglobin enzymatic hydrolysates and a significant amount of evidence has been accumulated indicating that the generation of these peptides (hemorphins) might occur in vivo. In order to investigate their putative physiological role and processing from hemoglobin in vivo, two methods were developed: a specific radioimmunoassay and a UV spectra comparison analysis. These methods were applied to a cathepsin D bovine hemoglobin hydrolysate and allowed the detection of two hemorphin-7 peptides. This observation supports the putative implication of cathepsin D in the in vivo release of hemorphins. Among the two methods used in this study, the immunological approach exhibits higher sensitivity and represents a useful method to investigate the in vivo role and physiological processing of hemorphins.     

乳杆菌胞外多糖合成基因簇及构效关系

肠道菌群稳态对维护人体的健康具有至关重要的作用。作为肠道天然微生物群“守卫兵”,益生菌参与改善体内微生态平衡。乳杆菌(Lactobacillus)是一种肠道益生菌的代表,其合成的胞外多糖(exopolysaccharide, EPS)既可通过增益肠道内其他益生菌的生长来优化肠道微生态,还具有抗肿瘤、抗氧化、降胆固醇、降血压和增强机体免疫力等益生功能。本文对近年来乳杆菌胞外多糖的遗传、生物学活性、构效关系等方面的研究进展,进行综述和展望。     

HORMAD2 methylation‐mediated epigenetic regulation of gene expression in thyroid cancer

This study is aimed to investigate the methylation level of candidate genes and its impact on thyroid carcinoma (THCA) development. Infinium Human Methylation 450 BeadChip Arrays by Illumina (Illumina HM450K) was the most popular CpG microarray platform widely used in biological and medical research. The methylation level of differentially expressed genes and their corresponding CpG sites were analysed by R programme. The expression of HORMAD2 was evaluated by qRT‐PCR and Western blot, while the methylation level was examined via methylation‐specific PCR. Cell viability, metastasis, cell cycle and apoptosis were detected by MTT assay, transwell and wound healing assay and flow cytometry, respectively, after treatment with 5‐aza‐2′‐deoxycytidine (5‐Aza). Tumour formation assay was used to analyse thyroid tumour growth in nude mice in vivo. The methylation levels of all 116 differentially expressed genes were analysed. HORMAD2 was significantly hypermethylated and its mRNA expression was inhibited in THCA cells. After treatment with 5‐Aza, HORMAD2 expression was up‐regulated in THCA cells and its overexpression can suppress thyroid cancer cell viability, mobility and invasiveness remarkably. Up‐regulation of HORMAD2 in THCA cells could prolong G0/G1 phase and shorten S phase to impede cell mitosis as well as promote thyroid cancer cells apoptosis. Furthermore, tumour formation assay showed that increased HORMAD2 level impeded tumour growth in vivo. Hypermethylation of HORMAD2 could induce THCA progression, while hypomethylation of HORMAD2 retard cell growth and mobility and facilitate apoptosis through increasing its mRNA expression.     

兴安、长白及华北落叶松RAPD 分子标记的物种特异性鉴定

以中国北方地区主要乡土落叶松树种兴安落叶松(Larix gmelini)、长白落叶松(L. olgensis)和华北落叶松(L. principisrupprechtii)的针叶及种子胚乳为研究材料, 采用RAPD分子标记技术对3种落叶松进行不同物种的种间鉴别。结果表明, 通过引物筛选得到了4个可以鉴别3种落叶松的RAPD引物, 其中有2个引物在落叶松针叶和种子胚乳基因组中都扩增出相同的条带。引物OPB-11在兴安和长白落叶松基因组DNA中1 500 bp处扩增出特异条带, 而在华北落叶松中没有; 引物OPX-14在兴安落叶松基因组DNA中1 200 bp处扩增出特异条带, 而在长白落叶松中没有; 还有2个引物可分别作为3种落叶松苗木和种子鉴别的辅助标记。本研究从分子水平上为落叶松的种间鉴别提供了新的鉴定方法。     

Expression of signaling molecules associated with apoptosis in human ischemic stroke tissue

There is growing evidence that, because of the highly significant differences in gene activation/protein expression between animal models of stroke and stroke patients, the current treatment strategies based on animal stroke models have been unsuccessful. Therefore, it is imperative that the pathobiology of human stroke be studied. As a first step here, Western blotting and immunohistochemistry were employed to examine expression and tissue localization of key apoptotic proteins in infarct and peri-infarcted (penumbra) from grey and white matter in human postmortem tissue of 18 patients who died between 2 and 37 d after stroke caused by large vessel disease. The contralateral hemisphere was used as a control. JNK1, JNK2, and p53 were upregulated in the majority of samples, whereas Bcl-2, caspase-3, active caspase-3, phosphorylated p53 (p-p53), phosphorylated JNk1 (p-JNK1), and phosphorylated JNK2 (p-JNK2) were upregulated in approximately half of the samples. JNK1 expression was positively correlated with JNK2 expression in grey and white matter infarct and penumbra, whereas active caspase-3 levels were positively correlated with p-JNK2 levels in grey and white matter infarct. Using indirect immunoperoxidase staining of paraffin-embedded sections, active caspase-3 was found in infarcted neurons that co-localized with TUNEL-positive cells. p-JNK localization in the nuclei of TUNEL-positive cells with the morphological appearance of neurons from infarct and penumbra was also demonstrated. The use of Kaplan Meier survival data demonstrated that the presence of Bcl-2 in penumbra of grey matter correlated significantly with shorter survival (p=0.006). In conclusion, the present study has identified significantly altered expression of apoptotic proteins in human stroke tissue and shown that the presence of Bcl-2 in penumbra of grey matter has prognostic value. It is tempting to suggest that further studies of apoptotic proteins in human stroke may lead to identification of novel targets for drug discovery.     

兴安、长白及华北落叶松RAPD分子标记的物种特异性鉴定

以中国北方地区主要乡土落叶松树种兴安落叶松（Larix gmelini）、长白落叶松（L.olgensis）和华北落叶松（L．principis—rupprechtii）的针叶及种子胚乳为研究材料,采用RAPD分子标记技术对3种落叶松进行不同物种的种间鉴别。结果表明,通过引物筛选得到了4个可以鉴别3种落叶松的RAPD引物,其中有2个引物在落叶松针叶和种子胚乳基因组中都扩增出相同的条带。引物OPB-11在兴安和长白落叶松基因组DNA中1500bp处扩增出特异条带,而在华北落叶松中没有：引物OPX-14在兴安落叶松基因组DNA中1200bp处扩增出特异条带,而在长白落叶松中没有：还有2个引物可分别作为3种落叶松苗木和种子鉴别的辅助标记。本研究从分子水平上为落叶松的种间鉴别提供了新的鉴定方法。     

Expression and characterization of full-length Ampullaria crossean endoglucanase EG65s and their two functional modules

Three endoglucanase cDNAs, eg65a, eg65b, and eg65c, were cloned from the mollusk Ampullaria crossean in previous work. To characterize the full-length enzymes as well as their individual functional modules via heterologous expression analysis, the three full-length putative endoglucanases (rEG65a, rEG65b, and rEG65c) and the corresponding catalytic modules (EG65a-CM, EG65b-CM, and EG65c-CM) were expressed in Pichia pastoris GS115, and the three corresponding carbohydrate-binding modules (EG65a-CBM, EG65b-CBM, and EG65c-CBM) were expressed in Escherichia coli BL21 (DE3). The properties of recombinant rEG65b, EG65a-CM, EG65b-CM, and EG65c-CM were characterized. Binding assays of CBMs with insoluble polysaccharides indicated that both EG65b-CBM and EG65c-CBM bound to phosphoric-acid swollen cellulose (PASC), Avicel, and oat-spelt xylan, while EG65a-CBM did not. The relative equilibrium constants (K(r)) of EG65b-CBM and EG65c-CBM were determined by absorption isotherm measurements. In this study, the CBMs of animal cellulases were expressed and characterized for the first time.