CHROMATIN REMODELING IN SPERMATIDS: A SENSITIVE STEP FOR THE GENETIC INTEGRITY OF THE MALE GAMETE

Several causes of male infertility remain idiopathic. Recently, the condensed state of the sperm head has been demonstrated as a discriminating parameter for the assessment of male fertility. Altered DNA condensation is associated with an increase in DNA strand breakage so the genetic integrity of the male gamete is threatened. The origin of the DNA strand breaks is unknown. However, transient DNA strand breaks appear in the whole population of elongating spermatids during mid-spermiogenesis steps. Most likely, these transient breaks are required to support the change in DNA topology associated with chromatin remodeling at these steps. Histones hyperacetylation is also coincident with the DNA strand breakage steps. Hyperacetylation of histones may represent a necessary condition for strand breakages to form allowing access to the yet unknown enzymatic activity involved in the removal of DNA supercoils. A better characterization of this enzyme activity at these steps is necessary as this may represent a very sensitive process where alterations in the genetic integrity of the male gamete may arise and persist up to the mature spermatozoa. During the chromatin remodeling in spermatids, the combined DNA-condensing activities provided by the basic transition proteins and protamines may optimize the strand repair process emphasizing the link between altered sperm DNA condensation and DNA fragmentation. The mutagenic potential of these events may have been overlooked as it may result in fertility and/or developmental problems.     

Icariin Improves Cognitive Impairment after Traumatic Brain Injury by Enhancing Hippocampal Acetylation

Objective

To examine the effect of icariin (ICA) on the cognitive impairment induced by traumatic brain injury (TBI) in mice and the underlying mechanisms related to changes in hippocampal acetylation level.

Methods

The modifified free-fall method was used to establish the TBI mouse model. Mice with post-TBI cognitive impairment were randomly divided into 3 groups using the randomised block method (n=7): TBI (vehicle-treated), low-dose (75 mg/kg) and high-dose (150 mg/kg) of ICA groups. An additional sham-operated group (vehicle-treated) was employed. The vehicle or ICA was administrated by gavage for 28 consecutive days. The Morris water maze (MWM) test was conducted. Acetylcholine (ACh) content, mRNA and protein levels of choline acetyltransferase (ChAT), and protein levels of acetylated H3 (Ac-H3) and Ac-H4 were detected in the hippocampus.

Results

Compared with the sham-operated group, the MWM performance, hippocampal ACh content, mRNA and protein levels of ChAT, and protein levels of Ac-H3 and Ac-H4 were signifificantly decreased in the TBI group (P<0.05). High-dose of ICA signifificantly ameliorated the TBI-induced weak MWM performance, increased hippocampal ACh content, and mRNA and protein levels of ChAT, as well as Ac-H3 protein level compared with the TBI group (P<0.05).

Conclusion

ICA improved post-TBI cognitive impairment in mice by enhancing hippocampal acetylation, which improved hippocampal cholinergic function and ultimately improved cognition.

     

LncRNA HULC降低PTEN甲基化及组蛋白乙酰化促进人胶质母细胞瘤细胞增殖和侵袭

目的:探讨长链非编码RNA肝癌高表达转录本（long non-coding RNA highly up-regulated in liver cancer,LncRNA HULC）降低PTEN启动子甲基化以及组蛋白乙酰化促进胶质母细胞瘤细胞增殖和侵袭的作用。方法:稳转HULC的胶质母细胞瘤细胞株U251,分为LncRNA HULC过表达组（HULC组）及空白对照组（vec组）。实时荧光定量PCR（qRT-PCR）检测各组HULC、PTEN表达水平。亚硫酸氢盐处理后测序（Bisulfite Sequencing PCR,BSP）检测PTEN启动子甲基化水平。蛋白免疫印迹（Western Blotting,WB）检测PTEN、组蛋白H3/H4、乙酰化组蛋白H3/H4表达水平。细胞增殖实验、平板克隆形成实验、划痕-愈合实验和Transwell实验分别检测各组细胞增殖、迁移和侵袭能力。结果:与vec组相比,HULC组PTEN启动子甲基化水平及组蛋白乙酰化水平较低（P＜0.05）,细胞的增殖、迁移和侵袭能力显著增高（P＜0.05）。结论:LncRNA HULC降低PTEN启动子甲基化和组蛋白乙酰化水平,促进胶质母细胞瘤细胞的增殖和侵袭。     

The histone acetyltransferase hMOF is frequently downregulated in primary breast carcinoma and medulloblastoma and constitutes a biomarker for clinical outcome in medulloblastoma

Loss of H4 lysine 16 (H4K16) acetylation was shown to be a common feature in human cancer. However, it remained unclear which enzyme is responsible for the loss of this modification. Having recently identified the histone acetyltransferase human MOF (hMOF) to be required for bulk H4K16 acetylation, here we examined the involvement of hMOF expression and H4K16 acetylation in breast cancer and medulloblastoma. Analysis of a recent mRNA expression profiling study in breast cancer (n = 100 cases) and an array-CGH screen in medulloblastomas (n = 102 cases), revealed downregulation in 40% and genomic loss in 11% of cases, respectively. We investigated hMOF protein expression as well as H4K16 acetylation in large series of primary breast carcinomas (n = 298) and primary medulloblastomas (n = 180) by immunohistochemistry. In contrast to nontransformed control tissues, significant fractions of both primary breast carcinomas and medulloblastomas showed markedly reduced hMOF mRNA and protein expression. In addition, hMOF protein expression tightly correlated with acetylation of H4K16 in all tested samples. For medulloblastoma, downregulation of hMOF protein expression was associated with lower survival rates identifying hMOF as an independent prognostic marker for clinical outcome in univariate as well as multivariate analyses.     

P38 MAPK信号通路在漆树酸改善苯肾上腺素诱导的小鼠心肌细胞肥大中的作用

目的:探讨P38丝裂原活化蛋白激酶(P38 mitogen-activated protein kinase,P38 MAPK)信号通路在漆树酸改善苯肾上腺素(phenylephrine,PE)诱导的小鼠心肌细胞肥大中的作用。方法:原代培养新生小鼠心肌细胞,PE诱导心肌细胞肥大。按随机数字表法将细胞分为空白对照组、溶剂对照组、PE组、PE+漆树酸组、PE+漆树酸+P38抑制剂组和PE+P38抑制剂组。收集干预48 h后的乳鼠心肌细胞进行以下检测:Western blot检测p-P38、第9位赖氨酸乙酰化的组蛋白H3(acetylated histone H3 at lysine 9,H3K9ac)和心房钠尿肽(atrial natriuretic peptide,ANP)的蛋白表达水平,免疫共沉淀(co-immunoprecipitation,Co-IP)法验证p-P38与H3K9ac蛋白之间的相互作用,RT-qPCR检测肌细胞增强因子2C(myocyte enhancer factor 2C,MEF2C)mRNA表达水平,免疫荧光染色观察小鼠心肌细胞表面积。结果:Western blot及RT-qPCR结果表明小鼠心肌细胞中p-P38和H3K9ac水平在PE组显著高于空白对照组(P<0.05),心脏核心转录因子MEF2C及心肌肥厚标志物ANP表达水平在PE组显著高于空白对照组(P<0.05);而在PE处理的心肌细胞中,P38抑制剂和漆树酸能够显著降低p-P38和H3K9ac水平,且MEF2C转录水平及ANP蛋白表达水平在PE+P38抑制剂组和PE+漆树酸组也较PE组显著降低(P<0.05);Co-IP结果表明p-P38与H3K9ac存在相互调控关系;免疫荧光结果表明,与空白对照组相比,PE组心肌细胞表面积显著增大(P<0.05),而P38抑制剂和漆树酸干预后能显著降低PE诱导的心肌细胞肥大(P<0.05)。结论:漆树酸能改善PE诱导的心肌细胞肥大,其作用机制可能与调节P38 MAPK信号通路介导的组蛋白乙酰化修饰失衡有关。     

Simvastatin functions as a heat shock protein 90 inhibitor against triple‐negative breast cancer

Acetylation plays an important role in regulating the chaperone activity of heat shock protein 90 (Hsp90) during malignant transformation through the stabilization and conformational maturation of oncogenic proteins. However, the functional acetylation sites, potential anticancer drug targets, are still emerging. We found that acetylation at K292 in Hsp90α is critical for the development and treatment of breast cancer. Acetylation at K292 not only augments the affinity of Hsp90 to ATP, cochaperones, and client proteins but it also promotes cancer cell colony formation, migration, and invasion in vitro as well as tumor growth in vivo. Importantly, K292‐acetylated Hsp90 has been validated as an exciting anticancer drug target by interfering with the complex formation between K292‐acetylated Hsp90 and cochaperone Cdc37, leading to diminishment of kinase client maturation and proteasome‐dependent degradation of kinase substrates. Furthermore, we showed that simvastatin prevented, whereas LBH589 promoted, the progression of Hsp90 chaperone cycling and client maturation, resulting in an increment of cell apoptosis by the combination of simvastatin and LBH589 in a mouse xenograft model. These data suggest that simvastatin is a novel Hsp90 inhibitor to disrupt the formation of the K292‐acetylated Hsp90/Cdc37 complex in triple‐negative breast cancer cells. The combination of simvastatin with LBH589 could be used as a novel therapeutic strategy for triple‐negative breast cancer.     

黄蜀葵茎叶多糖的乙酰化修饰及其免疫调节活性研究

目的?应用乙酰化修饰方法与技术进行黄蜀葵茎叶多糖的结构修饰,并评价其体外免疫调节活性,以期为黄蜀葵茎叶废弃物的资源化利用提供思路和科学依据。方法?采用水提醇沉法及DEAE-52离子交换法制备黄蜀葵茎叶粗多糖（SLAMP）和中性多糖（SLAMP-a）,应用乙酸酐法制备3种乙酰化修饰产物（SLAMP-a1、SLAMP-a2、SLAMP-a3）,且通过化学组成分析、柱前衍生HPLC法以及红外光谱法对多糖结构进行初步鉴定;此外,通过考察SLAMP-a及其3种乙酰化修饰产物对脾淋巴细胞体外增殖及RAW264.7释放NO的影响,评价四种多糖的免疫调节活性。结果?SLAMP-a总糖含量为99.76%,无体外免疫调节活性;3种修饰产物中,只有SLAMP-a1具有显著的刺激脾细胞增殖作用及激活RAW264.7产生NO。SLAMP-a1总糖含量为82.50%,取代度为0.62,主要由葡萄糖组成,且含有少量的甘露糖、半乳糖及阿拉伯糖。结论?黄蜀葵茎叶多糖SLAMP-a经乙酰化修饰后,可显著提高体外免疫调节活性,SLAMP-a1有望开发成免疫调节剂,且乙酰化修饰方法与技术可作为黄蜀葵茎叶多糖资源化利用的有效途径。