Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.     

Molecular cloning and sequence analysis of the sisomicin biosynthetic gene cluster from <Emphasis Type="Italic">Micromonospora inyoensis</Emphasis>

Micromonospora inyoensis produces sisomicin (Sm), an aminoglycoside antibiotic. The gene cluster of sisomicin biosynthesis spanning ca. 47 kb consists of 37 ORFs encoding various proteins for sisomicin biosynthesis, regulation, resistance and transport. The comparative genetic studies on the biosynthetic genes of sisomicin and gentamicin (Gm) reveal a similar biosynthetic route and provide a framework for the future biosynthetic studies. An erratum to this article can be found at     

Polyploidization and sexual dimorphism of floral traits in a subdioecious population of Dasiphora glabra

银露梅亚雌雄异株种群的多倍化和花特征性二态 性二态是性别分离植物的常见性状,对植物的雌性和雄性功能可能产生不同的影响。本文以青藏高原特有植物银露梅(Dasiphora glabra)为研究对象,利用银露梅同时存在两性、雌性和雄性植株的种群,探讨银露梅与传粉相关的花特征性二态现象及其对传粉者访问、花粉流和结实的影响。另外,本文还利用流式细胞仪检查了两性植株、雄性植株和雌性植株之间基因组大小的差异。研究结果表明：银露梅雌性植株和雄性植株数量约占种群中植株总数量的40%,表明银露梅两性植株数量多于雄性和雌性的植株数量。两性植株的花数量显著多于雄性和雌性植株的花数量。雄花的花大小显著大于雌花和两性花的花大小。雄花的花粉数量与两性花的花粉数量没有差异,但是雌花的胚珠数量显著高于两性花的胚珠数量。双翅目的蝇类是银露梅最主要的访花者,尽管访花者偏好访问更大的花,但是在花间连续访问对大花没有表现出偏好。模拟花粉流实验表明,两性植株和雄性植株的有效花粉转移都很低,从而导致较低的结实率。雌性植株和雄性植株的DNA含量约为两性植株的4倍,并且单性植株和两性植株之间相互授粉不能结实。以上结果表明,与两性花相比,雌花和雄花在与传粉相关的花特征二态性上分别在雌性和雄性功能上表现出优势,但这种优势在很大程度上被低效的花粉转移所掩盖。银露梅的雌性植株和雄性植株经历了多倍化事件,导致与两性植株之间存在繁殖隔离,从而维持了3种性别植株的共存。     

几种中药DNA提取方法的比较研究

以丹参、绞股蓝、三七为材料,分别采取CTAB法和SDS法提取基因组DNA,并通过紫外分光光度法和琼脂糖凝胶电泳对所提取的DNA样品进行检测,将它们在DNA产量、质量等方面的优缺点进行总结。结果表明,CTAB法能从丹参、绞股蓝中提取高质量的DNA,而三七的DNA更适合用SDS法提取。     

Astrocyte calcium microdomains are inhibited by Bafilomycin A1 and cannot be replicated by low-level Schaffer collateral stimulation in situ

Astrocyte Gq GPCR and IP₃ receptor-dependent Ca²⁺ elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called “microdomains” and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms. Here, we applied manipulations to increase or inhibit neuronal vesicular neurotransmitter release together with low-level stimulation of Schaffer collaterals in acute mouse hippocampal slices in an effort to determine the mechanisms underlying spontaneous astrocyte Ca²⁺ events. We found no significant change in spontaneous microdomain astrocyte Ca²⁺ elevations when neuronal action potentials were significantly enhanced or blocked. The astrocyte Ca²⁺ activity was also not affected by inhibitors of group I mGluRs. However, blockade of miniature neurotransmitter release using Bafilomycin A1 significantly reduced the frequency of microdomain astrocyte Ca²⁺ elevations. We then tested whether astrocyte Ca²⁺ microdomains can be evoked by low intensity SC stimulation. Importantly, microdomains could not be reproduced even using single, low intensity pulses to the SCs at a minimum distance from the astrocyte. Evoked astrocyte Ca²⁺ responses most often included the cell soma, were reduced by group I mGluR antagonists, and were larger in size compared to spontaneous Ca²⁺ microdomains. Overall, our findings suggest that spontaneous microdomain astrocyte Ca²⁺ elevations are not driven by neuronal action potentials but require quantal release of neurotransmitter which cannot be replicated by stimulation of Schaffer collaterals.     

Neuraminidase 1 deficiency attenuates cardiac dysfunction,oxidative stress,fibrosis, inflammatory via AMPK-SIRT3 pathway in diabetic cardiomyopathy mice

Diabetic cardiomyopathy (DCM) is associated with oxidative stress and augmented inflammation in the heart. Neuraminidases (NEU) 1 has initially been described as a lysosomal protein which plays a role in the catabolism of glycosylated proteins. We investigated the role of NEU1 in the myocardium in diabetic heart. Streptozotocin (STZ) was injected intraperitoneally to induce diabetes in mice. Neonatal rat ventricular myocytes (NRVMs) were used to verify the effect of shNEU1 in vitro. NEU1 is up-regulated in cardiomyocytes under diabetic conditions. NEU1 inhibition alleviated oxidative stress, inflammation and apoptosis, and improved cardiac function in STZ-induced diabetic mice. Furthermore, NEU1 inhibition also attenuated the high glucose-induced increased reactive oxygen species generation, inflammation and, cell death in vitro. ShNEU1 activated Sirtuin 3 (SIRT3) signaling pathway, and SIRT3 deficiency blocked shNEU1-mediated cardioprotective effects in vitro. More importantly, we found AMPKα was responsible for the elevation of SIRT3 expression via AMPKα-deficiency studies in vitro and in vivo. Knockdown of LKB1 reversed the effect elicited by shNEU1 in vitro. In conclusion, NEU1 inhibition activates AMPKα via LKB1, and subsequently activates sirt3, thereby regulating fibrosis, inflammation, apoptosis and oxidative stress in diabetic myocardial tissue.     

Green tea extract attenuates hepatic steatosis by decreasing adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice

Excess hepatic lipid accumulation and oxidative stress contribute to nonalcoholic fatty liver disease (NAFLD). Thus, we hypothesized that the hypolipidemic and antioxidant activities of green tea extract (GTE) would attenuate events leading to NAFLD. Obese mice (ob/ob; 5 weeks old, n=38) and their lean littermates (n=12) were fed 0%, 0.5% or 1% GTE for 6 weeks. Then, hepatic steatosis, oxidative stress and inflammatory markers were measured. Obese mice, compared to lean controls, had greater hepatic lipids and serum alanine aminotransferase (ALT). GTE at 1% lowered (P<.05) hepatic lipids and ALT in obese mice. The GTE-mediated attenuation in hepatic steatosis was accompanied by decreased mRNA expression of adipose sterol regulatory element-binding protein-1c, fatty acid synthase, stearoyl CoA desaturase-1, and hormone-sensitive lipase and decreased serum nonesterified fatty acid concentrations. Immunohistochemical data indicated that steatotic livers from obese mice had extensive accumulation of tumor necrosis factor-α (TNF-α), whereas GTE at 1% decreased hepatic TNF-α protein and inhibited adipose TNF-α mRNA expression. Hepatic total glutathione, malondialdehyde and Mn- and Cu/Zn-superoxide dismutase activities in obese mice fed GTE were normalized to the levels of lean littermates. Also, GTE increased hepatic catalase and glutathione peroxidase activities, and these activities were inversely correlated with ALT and liver lipids. Collectively, GTE mitigated NAFLD and hepatic injury in ob/ob mice by decreasing the release of fatty acids from adipose and inhibiting hepatic lipid peroxidation as well as restoring antioxidant defenses and decreasing inflammatory responses. These findings suggest that GTE may be used as an effective dietary strategy to mitigate obesity-triggered NAFLD.     

The release of cytochrome c and the regulation of the programmed cell death progress in the endosperm of winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under waterlogging

It has been shown in mammalian systems that the mitochondria can play a key role in the regulation of apoptosis by releasing intermembrane proteins (such as cytochrome c) into the cytosol. Cytochrome c released from the mitochondria to the cytoplasm activates proteolytic enzyme cascades, leading to specific nuclear DNA degradation and cell death. This pathway is considered to be one of the important regulatory mechanisms of apoptosis. Previous studies have shown that endosperm cell development in wheat undergoes specialized programmed cell death (PCD) and that waterlogging stress accelerates the PCD process; however, little is known regarding the associated molecular mechanism. In this study, changes in mitochondrial structure, the release of cytochrome c, and gene expression were studied in the endosperm cells of the wheat (Triticum aestivum L.) cultivar “huamai 8” during PCD under different waterlogging durations. The results showed that waterlogging aggravated the degradation of mitochondrial structure, increased the mitochondrial permeability transition (MPT), and decreased mitochondrial transmembrane potential (ΔΨm), resulting in the advancement of the endosperm PCD process. In situ localization and western blotting of cytochrome c indicated that with the development of the endosperm cell, cytochrome c was gradually released from the mitochondria to the cytoplasm, and waterlogging stress led to an advancement and increase in the release of cytochrome c. In addition, waterlogging stress resulted in the increased expression of the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT), suggesting that the mitochondrial permeability transition pore (MPTP) may be involved in endosperm PCD under waterlogging stress. The MPTP inhibitor cyclosporine A effectively suppressed cell death and cytochrome c release during wheat endosperm PCD. Our results indicate that the mitochondria play important roles in the PCD of endosperm cells and that the increase in mitochondrial damage and corresponding release of cytochrome c may be one of the major causes of endosperm PCD advancement under waterlogging.