HPLC法检测苦杏仁苷及其酶解产物

本文建立了一个快速高效的HPLC方法同时检测苦杏仁苷及其4个体外酶解产物,色谱分析系统为安捷伦液相色谱系统,自动进样器,迪马ODS C18色谱柱(250 mm×4.6 mm,5μm),柱温25℃,流动相30%甲醇,检测波长210 nm,进样量10μL。结果表明,苦杏仁苷、phenyl-(3,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-acetonitrile、野樱花苷、苯甲醛和杏仁腈的保留时间分别为5.087、13.836、16.357、22.775和3.307 min。该HPLC方法的重现性好,精确度和准确度高,当柱温低于30℃、pH值介于6.0~7.0时该方法结果稳定、重现性好。     

Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway

Definitive endoderm differentiation is crucial for generating respiratory and gastrointestinal organs including pancreas and liver. However, whether epigenetic regulation contributes to this process is unknown. Here, we show that the H3K27me3 demethylases KDM6A and KDM6B play an important role in endoderm differentiation from human ESCs. Knockdown of KDM6A or KDM6B impairs endoderm differentiation, which can be rescued by sequential treatment with WNT agonist and antagonist. KDM6A and KDM6B contribute to the activation of WNT3 and DKK1 at different differentiation stages when WNT3 and DKK1 are required for mesendoderm and definitive endoderm differentiation, respectively. Our study not only uncovers an important role of the H3K27me3 demethylases in definitive endoderm differentiation, but also reveals that they achieve this through modulating the WNT signaling pathway.     

ALDH2, a novel protector against stroke?

In a recent paper published in Cell Research, an association between expression of mitochondrial aldehyde dehydrogenase (ALDH2), a mitochondrial chaperon expressed in the brain, and the prevalence of stroke is revealed. This finding indicates that ALDH2 may serve as a potential endogenous neuroprotective target and a promising therapeutic strategy for the management of stroke.Stroke is one of the leading causes of death and a major reason of adult chronic disability as well as age-related cognitive decline and dementia¹. Ischemic stroke represents > 80% of all stroke incidences with the remaining 20% due to primary hemorrhage. Proper management of the conventional risk factors for stroke, such as high blood pressure, elevated blood cholesterol, cigarette smoking, carotid stenosis, diabetes mellitus and heart failure, may reduce the incidence of stroke only to a certain degree, suggesting the existence of undiscovered or undefined risk factors^1,2. The unidentified risk factors for stroke, in conjunction with unsatisfactory control of known risk factors (e.g., high cholesterol and hypertension), may explain the intimate clinical challenge or failure for stroke management. To this end, identification of novel risk factors may hold promises in the development of strategies for prevention and treatment of stroke. Ample evidence has implicated the importance of genetic predisposition in the onset and progression of stroke². More recently, genome-wide association study (GWAS) approach has transformed the genetics of many complex chronic diseases and is just beginning to impact the field of stroke³. Genetic variants predisposing to ischemic stroke have been revealed by GWAS, such as two loci associated with atrial fibrillation (PITX2 and ZFHX3) linked to cardioembolic stroke and a locus on chromosome 9p21 tied to large-vessel stroke^1,4,5. Nonetheless, the precise contribution of genetics to the etiology of stroke, in particular various stroke subtypes, remains somewhat elusive. Gene candidates that have been identified to be associated with stoke warrant further validation in a large independent data set to consolidate their causative role in the pathogenesis of stroke.In a recent paper published in Cell Research, Guo and colleagues performed an unbiased proteomic examination and unveiled a unique role of deficiency in mitochondrial aldehyde dehydrogenase (ALDH2), the so-called “facial flash gene” responsible for detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE), in the pathogenesis of stroke⁶. In their study, overexpression or activation of ALDH2 conferred neuroprotection through clearance of 4-HNE whereas ALDH2 knockdown mitigated the neuroprotective property of PKCɛ. The PKCɛ-ALDH2 pathway was shown to mediate neuroprotection offered by moderate ethanol intake. Serum 4-HNE levels were inversely correlated with lifespan and elevated plasma 4-HNE levels were observed for at least 6 months following stroke onset. Perhaps the most intriguing evidence is that much higher initial plasma 4-HNE levels were associated with development of stroke in an 8-year follow-up study. These findings favor a likely role of ALDH2 in the prevalence of stroke or stroke-prone subjects, and furthermore, its therapeutic potential as a target in the management of stroke (Figure 1).Open in a separate windowFigure 1Schematic diagram depicting the possible interplay between ischemic stroke and elevated serum 4-HNE levels. Serum 4-HNE levels positively correlates with stroke injury and remains elevated after stroke. Deficiency and activation of ALDH2 significantly accentuates and attenuates stroke-associated cerebral ischemia injury, respectively.ALDH2 is a human gene found on chromosome 12. All Caucasians are homozygous for ALDH2 while approximately 50% of Asians are heterozygous and possess only one normal copy of the ALDH2 gene and one mutant copy encoding an inactive mitochondrial isozyme⁷. A recent meta-analysis of GWAS identified a tight association between ALDH2 genetic mutation and elevated blood pressure, a known risk factor for stroke, in Asian decedents⁸. This is supported by the recent observation favoring a crucial role for ALDH2 in the regulation of cardiovascular homeostasis in diabetes, alcoholism, endoplasmic reticulum stress, arrhythmias and ischemia-reperfusion injury^9,10,11. Stroke is known to interrupt mitochondrial function and promote mitochondrial swelling and depolarization, leading to ultimate neuronal cell death¹². ALDH2 exerts a major role in aldehyde detoxification in mitochondria, and attenuates or ablates neuronal mitochondrial damage. Reactive aldehydes, including MDA, 4-HNE and 1-palmitoyl-2-oxovaleroyl phosphatidyl choline (POVPC), all of which are potential substrates for ALDH2, are elevated in ischemic stroke injury^1,12. Higher levels of 4-HNE and MDA were found in the serum of stroke-prone hypertensive rats compared with normotensive WKY rats⁶. Interestingly, only 4-HNE, but not MDA, was elevated in stroke-prone hypertensive rats compared with hypertensive rats, suggesting a role of 4-HNE as a possible biomarker for stroke.Given that approximately 40% of the East Asian population carries an ALDH2*2 mutant allele with dramatic reduction in ALDH2 enzymatic activity, the current observation suggest that ALDH2 mutation serves as a risk factor for stroke⁶. Unlike its reported role in the heart, little information is available for ALDH2 in the brain and cerebrovascular function. Like all animal studies, caution needs to be taken to translate experimental findings to a clinical setting. It is noteworthy that the animal outcome studies were performed at a relatively short period after stroke. A longer time window should be essential to the ultimate assessment of stroke injury. Further studies are needed to uncover the precise mechanism behind the regulation of ALDH2 in stroke.     

雪白睡莲花酚类成分研究

采用大孔树脂、硅胶、ODS RP-18和Sephadex LH-20柱从雪白睡莲的干燥花蕾中分离得到7个酚类成分,通过理化性质和波谱学方法鉴定化合物的结构,分别为:isostrictiniin(1)、老鹤草素(2)、鞣花酸(3)、短叶苏木酚(4)、Annulatin 3’-O-β-D-xyloside(5)、短叶苏木酚酸甲酯(6),1,2,3,4,6-五没食子酰基葡萄糖(7)。这些化合物均为首次从该植物中分离得到,化合物1,2,5和6首次从该属植物中分得。     

月季查尔酮合成酶基因片段的克隆及其表达分析

采用改良CTAB法从月季(Rosa hybrida)花瓣中成功提取出总RNA,进行反转录合成cDNA模板。根据GenBank已发表的其他植物CHS基因序列的保守结构域设计简并引物,采用同源克隆法进行克隆,获得RhCHS片段序列,并分析该片段的生物学信息,利用半定量RT-PCR对不同花发育时期进行表达分析。序列分析结果表明,克隆到的cDNA片段长度为860 bp,编码287个氨基酸残基,GenBank登录号KC145553。同源序列比对发现,月季RhCHS与其他植物的同源性很高,说明CHS在进化的过程中具有高度的保守性。表达谱分析表明,CHS在盛花期表达量最高。     

温度对亚热带地区常见树种叶片甲烷排放的影响

以亚热带常见树种米槠、木荷、浙江桂、罗浮栲、杉木和柑橘为对象,利用控制试验研究了温度对树木叶片甲烷(CH4)排放的影响.结果表明:当温度在10℃时,供试的6种树木中,仅木荷、柑橘和罗浮栲的叶片排放CH4;温度高于20℃时,所有树木叶片均可排放CH4.温度高于30℃时,叶片排放CH4的平均排放速率(1.010ngCH4·g-1DM·h-1)是10～30℃时平均排放速率(0.255ngCH4·g-1DM·h-1)的3.96倍.增温对柑橘和杉木CH4排放速率的影响显著高于其他4种树木.培养时间对叶片排放CH4速率有显著影响,温度胁迫对树木排放CH4的影响受植物活性的控制.在低温或高温条件下,树木干叶均不能排放CH4.高温胁迫对树木叶片排放CH4有重要影响,全球变暖可能增加植物的CH4排放.     

两种玻璃化胚胎冷冻方法对不同品系小鼠胚胎冷冻效果比较

目的探讨EFS和DAP两种玻璃化冷冻方法对不同品系小鼠胚胎冷冻的效果。方法6个品系小鼠（KM、ICR、BALB／c、C57BL／6J、OB／OB、LAP／~TAOF59）的2-cell胚胎分别用EFS和DAP两种玻璃化冷冻方法进行冷冻和复苏,比较两种冷冻方法的胚胎复苏率和着床率。结果6个品系小鼠冷冻胚胎EFS方法的平均复苏率为69．97％（47．9％～83．6％）,DAP方法的平均复苏率47．23％（26．3％-76．7％）,EFS方法明显优于DAP方法。其中KM、ICR和BALB／c小鼠EFS方法的冷冻复苏率显著高于DAP方法（P〈0．01）;冻融胚胎移植后EFS方法的平均着床率27．23％（1．75％一45．0％）,DAP方法的平均着床率31．43％（7．0％一46．3％）。除KM、ICR小鼠外,其他4个品系小鼠的着床率DAP方法高于EFS方法。结论KM和ICR远交群小鼠胚胎适合用EFS方法冷冻保存;C57BL／6J、OB／OB、LAP／aTAOF59三个品系小鼠DAP方法优于EFS方法,但差异不大;BALB／c小鼠两种玻璃化冷冻方法的冻融胚胎着床率均较低,需进一步研究。     

Influence of gestational overfeeding on myocardial proinflammatory mediators in fetal sheep heart

Maternal overnutrition is associated with predisposition of offspring to cardiovascular disease in later life. Since maternal overnutrition may promote fetal and placental inflammatory responses, we hypothesized that maternal overnutrition/obesity increases expression of fetal cardiac proinflammatory mediators and alter cardiac morphometry. Multiparous ewes were fed either 150% of National Research Council (NRC) nutrient recommendations (overfed) or 100% of NRC requirement (control) from 60 days prior to mating to gestation Day 75 (D75), when ewes were euthanized. An additional cohort of overfed and control ewes were necropsied on D135. Cardiac morphometry, histology, mRNA and protein expression of toll-like receptor 4, iNOS, IL-1a, IL-1b, IL-6, IL-18, CD-14, CD-68, M-CSF and protein levels of phosphorylated I-κB and nuclear factor κB (NF-κB) were examined. Immunohistochemistry was performed to assess neutrophil and monocyte infiltration. Crown rump length, left and right ventricular free wall weights as well as left and right ventricular wall thickness were significantly increased in D75 fetuses of overfed mothers. Hematoxylin and eosin staining revealed irregular myofiber orientation and increased interstitial space in fetal ventricular tissues born to overfed mothers. Oil red O staining exhibited marked lipid droplet accumulation in the overfed fetuses. Overfeeding significantly enhanced TLR4, IL-1a, IL-1b IL-6 expression, promoted phosphorylation of IκB, decreased cytoplasmic NF-κB levels and increased neutrophil and monocyte infiltration. Collectively, these data suggest that maternal overfeeding prior to and throughout gestation leads to inflammation in the fetal heart and alters fetal cardiac morphometry.     

A peptide receptor-based bioelectronic nose for the real-time determination of seafood quality

We herein report a peptide receptor-based bioelectronic nose (PRBN) that can determine the quality of seafood in real-time through measuring the amount of trimethylamine (TMA) generated from spoiled seafood. The PRBN was developed using single walled-carbon nanotube field-effect transistors (SWNT-FETs) functionalized with olfactory receptor-derived peptides (ORPs) which can recognize TMA and it allowed us to sensitively and selectively detect TMA in real-time at concentrations as low as 10fM. Utilizing these properties, we were able to not only determine the quality of three kinds of seafood (oyster, shrimp, and lobster), but were also able to distinguish spoiled seafood from other types of spoiled foods without any pretreatment processes. Especially, the use of small synthetic peptide rather than the whole protein allowed PRBNs to be simply manufactured through a single-step process and to be reused with high reproducibility due to no requirement of lipid bilayers. Furthermore, the PRBN was produced on a portable scale making it effectively useful for the food industry where the on-site measurement of seafood quality is required.