Shi-pi-xiao-ji formula suppresses hepatocellular carcinoma by reducing cellular stiffness through upregulation of acetyl-coA acetyltransferase 1

BACKGROUND Previous studies have shown that the Shi-pi-xiao-ji(SPXJ)herbal decoction formula is effective in suppressing hepatocellular carcinoma(HCC),but the underlying mechanisms are not known.Therefore,this study investigated whether the antitumor effects of the SPXJ formula in treating HCC were mediated by acetyl-coA acetyltransferase 1(ACAT1)-regulated cellular stiffness.Through a series of experiments,we concluded that SPXJ inhibits the progression of HCC by upregulating the expression level of ACAT1,lowering the level of cholesterol in the cell membrane,and altering the cellular stiffness,which provides a new idea for the research of traditional Chinese medicine against HCC.AIM To investigate the anti-tumor effects of the SPXJ formula on the malignant progression of HCC.METHODS HCC cells were cultured in vitro with SPXJ-containing serum prepared by injecting SPXJ formula into wild-type mice.The apoptotic rate and proliferative,invasive,and migratory abilities of control and SPXJ-treated HCC cells were compared.Atomic force microscopy was used to determine the cell surface morphology and the Young’s modulus values of the control and SPXJ-treated HCC cells.Plasma membrane cholesterol levels in HCC cells were detected using the Amplex Red cholesterol detection kit.ACAT1 protein levels were estimated using western blotting.RESULTS Compared with the vehicle group,SPXJ serum considerably reduced proliferation of HCC cells,increased stiffness and apoptosis of HCC cells,inhibited migration and invasion of HCC cells,decreased plasma membrane cholesterol levels,and upregulated ACAT1 protein levels.However,treatment of HCC cells with the water-soluble cholesterol promoted proliferation,migration,and invasion of HCC cells as well as decreased cell stiffness and plasma membrane cholesterol levels,but did not alter the apoptotic rate and ACAT1 protein expression levels compared with the vehicle control.CONCLUSION SPXJ formula inhibited proliferation,invasion,and migration of HCC cells by decreasing plasma membrane cholesterol levels and altering cellular stiffness through upregulation of ACAT1 protein expression.

Histone acetyltransferase GCN5 interferes with the miRNA pathway in Arabidopsis

MicroRNAs （miRNA） that guide sequence-specific posttranscriptional gene silencing play an important role in gene expression required for both developmental processes and responses to environmental conditions in plants. However, little is known about the transcriptional and posttranscriptional regulation of miRNA expression. Histone acetylation plays an important role in chromatin remodeling and is required for gene activation. By analyzing the accumulation of subset of miRNAs and the corresponding primary miRNAs in mutants of Arabidopsis, we show that histone acetyltransferase GCN5 （General control non-repressed protein 5） has a general repressive effect on miRNA production, while it is required for the expression of a subset of （e.g. stress-inducible） MIRNA genes. The general negative function of GCN5 in miRNA production is likely achieved through an indirect repression of the miRNA machinery genes such as DICER LIKE1 （DCL1）, SERRATE （SE）, HYPONASTIC LEAVES1 （HYL1） and ARGONAUTE1 （AGO1）. Chromatin immunoprecipitation assays revealed that GCN5 targets to a subset of MIRNA genes and is required for acetylation of histone H3 lysine 14 at these loci. Moreover, inhibition of histone deacetylation by trichostatin A treatment or in histone deacetylase gene mutants impaired the accumulation of certain miRNAs. These data together suggest that Arabidopsis GCN5 interferes with the miRNA pathway at both the transcriptional and posttranscriptional levels and histone acetylation/deacetylation is an epigenetic mechanism involved in the regulation of miRNA production.

Effect of Nao Yikang on choline acetyltransferase and caspase-3 brain expression in a rat model of Alzheimer's disease

BACKGROUND： The main components of the traditional Chinese medicine compound Nao Yikang have been shown to possibly alleviate neural damage. OBJECTIVE： To observe the effects of Nao Yikang on expression of choline acetyltransferase （CHAT） and caspase-3 in the rat brains of an experimental Alzheimer＇s disease （AD） model, and to investigate the mechanisms of potential neuroprotective effects. DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Department of Pathophysiology, Medical School of Nantong University between November 2006 and December 2007. MATERIALS： The main active components of Nao Yikang were as follows： prepared polygonum multiflorum, Rhizoma anemarrhenae, and Rhizoma acori tatarinowii. Nao Yikang granules were prepared by Nantong Hospital of Traditional Chinese Medicine. Ibotenic acid （IBO） was purchased from Sigma-Aldrich, USA, ChAT goat anti-rat antibody from Chemicon, USA, and cleaved caspase-3 rabbit anti-rat （Asp175） （5A1） antibody from Cell Signaling, USA. METHODS： A total of 60 male, Sprague Dawley rats （2 months old） were randomly assigned to 6 groups： sham-surgery, model, Nao Yikang 1.73, 3.45, 6.90 g/kg per day, and piracetam, with 10 rats in each group. Bilateral infusions of 5 pg IBO into the nucleus basalis of Meynert were performed with Hamilton syringe and stereotaxic apparatus for AD model establishment. For the sham-surgery group, rats received 1 μL saline in the identical stereotaxic position. From the second day, Nao Yikang groups were administrated 1.73, 3.45, and 6.90 g/kg per day Nao Yikang, respectively, while the piracetam group received 0.04 g/mL piracetam, the model group received 0.5% sodium carboxymethyl cellulose, and the sham-surgery group received normal saline. Rats were intragastrically administered 1 mL/100 g daily for 28 consecutive days. MAIN OUTCOME MEASURES： Following treatment of the various solutions for 28 days, Western blot was utilized to observe ChAT expression in the frontal cortex of AD rats, and immunohistochemistry was applied to quantify caspase-3-positive cells in the frontal cortex. RESULTS： ChAT protein expression significantly decreased in the model group （P 〈 0.01）, however caspase-3 expression was significantly elevated （P 〈 0.01） compared with the sham-surgery group. Compared with the model group, ChAT protein expression increased in the Nao Yikang 1.73 g/kg per day, 3.45 g/kg per day, 6.90 g/kg per day groups, and the piracetam group （P 〈 0.05 or P 〈 0.01） and the number of caspase-3-positive cells decreased in the Nao Yikang 3.45 g/kg per day and 6.90 g/kg per day groups （P 〈 0.01）. However, there was no change in the number of caspase-3-positive cells in the 3.45 g/kg per day group. CONCLUSION： The traditional Chinese medicine compound Nao Yikang increased ChAT protein expression and suppressed caspase-3 expression in the frontal cortex in a dose-dependent manner.

Estrogen intervention in microvascular morphology and choline acetyltransferase expression in rat hippocampal neurons in chronic cerebral ischemia

We observed dynamic changes in microvessels and a protective effect of estrogen on chronic cerebral ischemia ovariectomized rat models established through permanent occlusion of bilateral carotid arteries at 7, 14 and 21 days. The results revealed that estrogen improved microvasculature in the hippocampus of chronic cerebral ischemic rats, upregulated Bcl-2 protein expression, downregulated Bax protein expression, increased choline acetyltransferase expression in hippocampal cholinergic neurons, and suppressed hippocampal neuronal apoptosis. These findings indicate that estrogen can protect hippocampal neurons in rats with chronic cerebral ischemia.

Polymorphism of N-acetyltransferase 2 (NAT2) Gene Polymorphism in Shanghai population: Occupational and Non-occupational Bladder Cancer Patient Groups

Objective Arylamine N-acetyltransferases (NATs) are involved in the detoxification of aromatic amines and hydrazine. In order to explore the possible association of NAT2 polymorphism with bladder cancer risk in benzidine exposed or non-exposed Chinese individuals, healthy subjects, subjects with bladder cancer of a former benzidine exposed cohort in Shanghai dyestuff industry and a group of bladder cancer patients without known occupational exposure to aromatic amines were genotyped for NAT2 gene polymorphism. Methods NAT2 genotyping was performed with a set of RFLP procedures at seven major polymorphic loci of gene coding area: G191A, C282T, T341C, C481T, G590A, A803G and G857A. Results The wild allele NAT2 *4 was the most prevalent allele (59%) in healthy individuals. The alleles NAT2*6A and NAT2*7B were also frequently observed (21% and 17%, respectively). In contrast to Caucasians, the percentage of slow acetylators was lower (12% in Chinese vs. 58% in Caucasians, P<0.001). No relevant differences were observed for homogenous rapid, heterogeneous rapid/slow and homogeneous slow acetylation genotypes between the healthy subjects and both groups of bladder cancer patients. Conclusion The present work did not support the association of slow acetylating genotypes of NAT2 gene with elevated risk of bladder cancer in Chinese whereas it was documented as an important genetically determined risk factor in Caucasians. Different mechanisms might play a role in individual susceptibility to bladder cancer related with aromatic amine exposure in various races or ethnic groups.

Effects of gentamicin on choline acetyltransferase expression in paraolivary nucleus neurons of guinea pigs

BACKGROUND： It is generally accepted that gentamicin can damage the cochlear nerve and acoustic nerve. In recent years, scholars have focused on neuronal changes and neurochemical information in the brainstem primary auditory center. OBJECTIVE： To explore morphological changes of choline acetyltransferase （ChAT）-positive neurons in the paraolivary nucleus （PON） of guinea pigs, and the effect on hearing following gentamicin injection. DESIGN, TIME AND SETTING： Randomized grouping and morphological observational study was performed at Animal Experimental Center of General Hospital of Shenyang Military Area Command of Chinese PLA from January to August 2007. MATERIALS： A total of 48 healthy guinea pigs were randomly divided into model （n = 40） and control （n = 8） groups. The model group was divided into five subgroups at five time points of 1 and 3 days, 1, 2, and 3 weeks. METHODS： Guinea pigs in the model group were intraperitoneally injected with gentamicin, and those in the control group were intraperitoneally injected with the same volume of saline. MAIN OUTCOME MEASURES： Auditory brainstem-evoked potential was used to record auditory threshold; distribution and morphological changes of ChAT-positive neurons in the PON were observed with immunohistochemistry; section area and gray value of ChAT-positive neurons were measured with Quantimet 570 image-analyzing system. RESULTS： ChAT-positive neurons were diffusedly distributed in the PON. The majority was composed of large, round cells, with positive neurites that could be clearly observed. Following gentamicin injection, the positive neurons displayed an irregular outline, and their neurites began to shorten and disappear. The gray value increased with prolonged gentamicin administration （P 〈 0.05）. In addition, the somatic cross-sectional area was enlarged in the model group at 1 and 3 days after injection （P 〈 0.05）, whereas cell number significantly decreased at ;three weeks after injection （P 〈 0.05）. Starting at 3-4 days, behavioral features and auditory degrees became gradually aggravated with prolonged gentamicin administration （P 〈 0.05）. CONCLUSION： Gentamicin damaged ChAT-positive neurons in the PON, and long-term gentamicin treatment aggravated hearing impairment.

Choline acetyltransferase expressed by radial neuroglia cells in the development of telencephalon: A validated study

BACKGROUND： Cholinergic neuron directly participants in human motion, learning and memory and is a target cell for multiple degenerative diseases of central nervous system. OBJECTIVE： To investigate whether the mitotic cell is the radial glial cell expressing choline acetyltransferase （CHAT） in ventricle zone （VZ） of telencephalon and whether cholinergic neuron is derived from radial glial cell in ventricle zone of telencephalon. DESIGN： Observational study. SETTING： Department of Histology and Embryology, Basic Medical College of Jilin University. MATERIALS： Nine healthy Wistar rats included 6 females and 3 male. Male and female rats were mated routinely, and the day when spermatozoa or vaginal plug were found was regarded as embryonic 0 （E0）. Primary monoclonal antibodies ChAT and vimentin were provided respectively by Wuhan Boster Company, and Biogenex Company, USA. METHODS： The experiment was carried out in the Laboratory of Cell Culture and Immunohistochemistry, Department of Histology and Embryology from march 2002 to January 2003. Firstly, fluorescence-activated cell sorting （FACS） was used to confirm the time of generation of cholinergic neuron; secondly, telencephalons of rats at embryonic 14 days （E14） were performed coronary sections, then immunohistochemistry double staining for vimentin （a protein marker of radial neuroglia cell） and ChAT （a protein marker of cholinergic neuron） were used to test whether ChAT was expressed in the radial neuroglia cells. MAIN OUTCOME MEASURES： （1） Fluorescence-activated cell numbers of ChAT in telencephalon; （2） results of immunohistochemistry double staining. RESULTS： It is confirmed using by flow cytometer that embryogenesis time of cholinergic neuron was at E12, and shown the population of cells in VZ of dorsal telencephalon of E14 rat co-expressed vimentin and ChAT through immunohistochemistry double staining. A lot of vimentin-positive cells and ChAT-positive cells respectively were observed in VZ of lateral ganglionic eminence. CONCLUSION： Cholinergic neuron in cerebral cortex is derived from radial glial cells in VZ of dorsal telencephalon; meanwhile, cholinergic neuron of striatum is derived from radial glial cells in VZ of lateral ganglionic eminence.

Submicroscopic 11p13 deletion including the elongator acetyltransferase complex subunit 4 gene in a girl with language failure, intellectual disability and congenital malformations: A case report

BACKGROUND We described the main features of an infant diagnosed with facial dysmorphic,language failure,intellectual disability and congenital malformations to strengthen our understanding of the disease.Currently,treatment is only rehabilitation and surgery for cleft lip and palate.CASE SUMMARY The proband was a 2-years-8-months-old girl.Familial history was negative for congenital malformations or intellectual disability.The patient had microcephaly,upward-slanting palpebral fissures,depressed nasal bridge,bulbous nose and bilateral cleft lip and palate.Brain magnetic resonance imaging showed cortical atrophy and band heterotopia.Her motor and intellectual development is delayed.A submicroscopic deletion in 11p13 involving the elongator acetyltransferase complex subunit 4 gene(ELP4)and a loss of heterozygosity in Xq25-q26.3 were detected.CONCLUSION There is no treatment for the ELP4 deletion caused by a submicroscopic 11p3 deletion.We describe a second case of deletion of the ELP4 gene without aniridia,which confirms the association between ELP4 gene with several defects and absence of this ocular defect.Additional clinical data in the deletion of the ELP4 gene as cleft palate,facial dysmorphism,and changes at level brain could be associated to this gene or be part of the effect of the recessives genes involved in the loss of heterozygosity region of Xq25-26.3.

Bifurcation Analysis of a Coupled Acetylcholinesterase/Choline Acetyltransferase Enzymes Neurocycle

A diffusion-reaction, two-compartment model was used to explore the bifurcation and chaotic behavior of acetylcholinesterase (AChE) and cholineacetyltransferase (ChAT) coupled enzymes system. The effects of hydrogen ion feed concentrations, choline (Ch) and acetylcholine (ACh) feed concentrations, as bifurcation parameters on the system performance are studied. It is found that hydrogen ions play an important role in creating potential differences through the plasma membranes. Detailed bifurcation analysis over a wide range of parameters is carried out in order to uncover some of the qualitative changes of the system such as hysteresis, multiplicity, Hopf bifurcation, boundary crises bifurcation, periodic transient, and other complex dynamics. Some of the obtained results relate to the phenomena occurring in the physiological experiments like periodic stimulation of neural cells and irregular functioning of acetylcholine receptors. The model depends on real kinetics expressions and parameters obtained from the literature, so the results can be used to direct more systematic research on cholinergic disorder.

Acetyltransferase P300 Inhibits the Proliferation, Invasion, and Migration of Esophageal Cancer via Survivin Acetylation

Background: Esophageal cancer is one of the primary death causes leading by cancer in the world, which is high morbidity and mortality. Epigenetic acetylation modification participates in and regulates the proliferation, invasion, and metastasis of various tumor cells, and the acetylation modification of tumor proteins involved by acetyltransferases may be one of the important mechanisms of esophageal carcinogenesis. The aim of this study was to investigate the correlation of acetyltransferase P300 and Survivin acetylation in esophageal cancer pathogenesis and its molecular mechanism. Methods: Fifty-five cases of esophageal cancer tissues and adjacent cancer tissues were collected, Survivin and P300 protein expression was measured by immunohistochemistry (SP) and protein blotting (Western Blot);Survivin acetylated protein levels were measured by coimmunoprecipitation (Co-IP);bioinformatics predicted the relationship between P300 and Survivin as the substrate, and fluorescence immunohistochemistry (IF) to verify the localization and expression of Survivin and P300 in esophageal cancer tissues;the correlation of Survivin acetylation, P300 and clinical cases characteristics was analyzed by statistics. P300 siRNA sequences were structured and transfected into EC109 cells. P300 protein expression and Survivin acetylated protein levels were determined by Co-IP. Cell viability was determined by the MTT assay, Scratch healing and Transwell chamber assay examined cell migration and invasion ability. Results: Survivin and P300 protein expression was significantly increased in human esophageal cancer tissues and EC109 cells. The Survivin protein was acetylated in esophageal cancer tissues and EC109 cells, and its protein acetylation rate was significantly increased;bioinformatics predicted that the acetyltransferase P300 could catalyze the acetylation of Survivin as a substrate, and the fluorescence immunohistochemistry confirmed that both Survivin and P300 simultaneously showed a high expression state in cancer tissues;Survivin acetylation and P300 expression;Survivin acetylation and P300 were closely related with esophageal cancer stage, tissue differentiation and lymph node metastasis. The vitro experiments showed that P300 RNA interference in esophageal cancer cells can significantly reduce the Survivin protein acetylation level, while inhibiting the survival, migration and invasion capacity of EC109 cells. Conclusion: P300 has a correlation with Survivin acetylation in the lespathological process of esophageal cancer, P300 may be an important upstream molecule of Survivin acetylation and has an important potential value in the diagnosis and treatment of esophageal cancer.

Arylamine N-acetyltransferases: a new inhibitor of apoptosis in HepG2 cells

Objective: To explore how arylamine N-acetyltransferases (NATs) is related to cell apoptosis. Methods: NAT activity in apoptotic HepG2 cells was measured using high performance liquid chromatography (HPLC); the apoptosis rate of HepG2 cells acted upon by an NAT inhibitor was measured using flow cytometry. Results: NAT activity was lowered in apoptotic HepG2 cells; apoptosis rate induced by camptothecin (CAM) increased after inhibition of NAT activity in HepG2 cells. Conclusion: NAT can inhibit apoptosis in HepG2 cells.

N-acetyltransferase 2: Slow, intermediate or fast? A booming question of the molecular epidemiology in cancer research

Throughout history, humanity has referred to reactions occurring with food, plants and, recently, medicines or drugs. The increase in pulmonary tuberculosis cases and the availability of treatment showed that genetic human differences can interfere in the capacity to metabolize drugs. There are remarkable genetic polymorphisms of N-acetyltransferase 2 (NAT2) activity that have been associated with different levels of susceptibility to developing many kinds of cancers. This review considers the field as an open window for the application of molecular epidemiology tools that led to the development of pharmacogenomics. We cover historical data and the most recent knowledge about NAT2 genetic polymorphisms and its distribution in different populations, which is an important concept being incorporated in epidemiological studies of cancer risk. We present up to date information about these studies, including meta-analysis based on the NAT2 distribution in different types of cancer. A critical broad at advances in NAT2 research, high-lighting recent studies related to NAT2 alleles in cancer susceptibility. Although there are multifactorial aspects involved in cancer risk, the variability in NAT2 allelic frequency can be related to carcinogenesis through alterations in the metabolic rate after exposure to carcinogens.

Density Functional Theory Study on the Histidine-assisted Mechanism of Arylamine N-Acetyltransferase Acetylation

Arylamine N-acetyltransferases （NATs, EC 2.3.1.5） catalyze the N-acetylation of primary arylamines, and play a key role in the biotransformation and metabolism of drugs, carcinogens, etc. In this paper, three possible reaction mechanisms are investigated and the results indicate that if the acetyl group directly transfers from the donor to the acceptor, the high activation energies will make it hard to obtain the target products. When using histidine to mediate the acetylation process, these energies will drop in the 15-45 kJ/mol range. If the histidine residue is protonated, the corresponding energies will be decreased by about 35-87 kJ/mol. The calculations predict an enzymatic acetylation mechanism that undergoes a thiolate-imidazolium pair, which agrees with the experimental results very well.

胰淀素作用于背外侧被盖核胆碱乙酰转移酶神经元调节小鼠体重

胰淀素(amylin)作用于背外侧被盖核(lateral dorsal tegmental nucleus,LDT),诱导减肥和抑食效应,并激活背肩胛棕色脂肪组织(interscapular brown adipose tissue,IBAT)产热。多种食欲激素受体与胆碱乙酰转移酶(choline acetyltransferase,ChAT)神经元共同定位于LDT中,以调节饱腹感和能量消耗。然而,LDT的ChAT神经元是否参与胰淀素介导的体重调节作用尚待探索。作者拟通过免疫荧光检测小鼠LDT中原癌基因蛋白(c-Fos)和ChAT神经元的表达,逆行示踪技术确认LDT的下游神经核团,神经元损伤试验分析胰淀素作用的神经机制。结果显示:腹腔注射胰淀素显著降低小鼠体重,并显著促进LDT和臂旁核(PBN)中c-Fos和ChAT神经元表达。ChAT和神经型一氧化氮合成酶(nNOS)神经元在LDT、PBN和迷走神经背侧核(DMV)中共表达。逆行示踪试验证明LDT向PBN神经元发送神经投射。此外,损伤LDT和PBN神经元可降低胰淀素的减肥效果。LDT是连接PBN的能量平衡调节中枢,ChAT神经元参与介导胰淀素的体重调节过程。

ACAT1和MTNR1B基因多态性与非酒精性脂肪性肝病易感性的关系

目的本研究拟探讨乙酰辅酶A乙酰转移酶1(ACAT1)和褪黑激素受体1B(MTNR1B)基因多态性与非酒精性脂肪性肝病(NAFLD)疾病易感性的关系。方法本研究共纳入2020年12月—2022年6月就诊于青岛市市立医院的健康体检者164例、NAFLD患者228例。采用PCR及测序的方法对ACAT1 rs1044925、rs1157651和MTNR1B rs10830963基因多态性进行基因分型,并采集空腹静脉血进行生化检测。符合正态分布的计量资料两组间比较采用成组t检验;非正态分布的计量资料两组间比较采用Mann-Whitney U非参数检验;计数资料两组间比较采用χ2检验。结果ACAT1 rs1044925、rs1157651和MTNR1B rs10830963基因型分布在NAFLD及健康对照组间无统计学差异(P值均>0.05),ACAT1 rs1044925 AA基因型携带者的LDL水平明显高于C等位基因携带者(Z=−2.08,P=0.04),MTNR1B rs10830963 G等位基因携带者空腹血糖水平明显高于CC基因型携带者(Z=−3.01,P<0.01)。结论ACAT1 rs1044925、rs1157651和MTNR1B rs10830963多态性与NAFLD易感性无明显相关,ACAT1 rs1044925和MTNR1B rs10830963位点分别与LDL和空腹血糖水平有关。

硫醇乙酰基转移酶mshD对结核分枝杆菌生长和应对压力刺激的研究

目的:探索硫醇乙酰基转移酶MshD对结核分枝杆菌在体外生长、应对压力刺激和活性氧(reactive oxygen species,ROS)水平的影响。方法:利用CRISPR-NHEJ基因编辑技术,构建结核分枝杆菌mshD基因敲除株(ΔmshD株)和回补株(ΔmshD::mshD株)。分别检测H37Rv野生株(WT)、ΔmshD株和ΔmshD::mshD株在液体培养基和固体培养基中的生长情况,以及外源添加L-半胱氨酸和过氧化氢酶对菌株生长的影响;检测3种菌株对不同刺激剂如H 2O 2、二硫苏糖醇(DTT)、十二烷基硫酸钠(SDS)的敏感性,以及外源添加过氧化氢酶对压力条件处理后菌株的恢复情况;采用流式细胞术检测SDS处理3种菌株前后菌株的ROS水平。结果:与WT和ΔmshD::mshD株相比,ΔmshD株在固体平板上的生长较为缓慢,外源添加过氧化氢酶可以恢复其生长趋势;ΔmshD株应对DTT[WT(6.96±2.02)%,ΔmshD(0.02±0.00)%,ΔmshD::mshD(6.64±0.77)%;F=29.700,P<0.001],H 2O 2[WT(0.23±0.06)%,ΔmshD(0.01±0.00)%,ΔmshD::mshD(0.26±0.06)%;F=25.520,P=0.001]和SDS[WT(0.12±0.03)%,ΔmshD(0.01±0.00)%,ΔmshD::mshD(0.18±0.04)%;F=19.540,P=0.002]刺激的存活率更低,差异均有统计学意义;外源添加过氧化氢酶可以恢复其存活率。ΔmshD株自身ROS水平高于WT(WT:95.100±2.553,ΔmshD:106.000±4.000,ΔmshD::mshD:94.667±3.055;F=11.650,P=0.009),SDS处理ΔmshD株的自身ROS水平进一步升高(WT:436.000±8.000,ΔmshD:533.667±4.726,ΔmshD::mshD:441.333±2.517;F=292.900,P<0.001),差异均有统计学意义。结论:mshD基因缺失在固体培养基中生长减慢,mshD基因协助结核分枝杆菌抵抗各种应激压力,ΔmshD菌株内源性ROS水平增加。外源添加过氧化氢酶可一定程度恢复mshD基因敲除株生长和存活缺陷。

P300低表达食管癌细胞株TE-1的Survivivn蛋白乙酰化、增殖迁移侵袭能力变化及其机制

目的观察乙酰基转移酶P300低表达食管癌细胞株TE-1细胞中的Survivivn蛋白乙酰化、增殖迁移侵袭能力变化,并探讨其机制。方法取对数生长期TE-1细胞分为si-P300组、si-P300-NC组、NC组。si-P300组采用脂质体介导法转染乙酰基转移酶P300下调质粒,si-P300-NC组采用脂质体介导法转染阴性对照质粒,NC组不转染质粒,转染后继续孵育48 h进行后续实验。取各组细胞,采用免疫共沉淀(Co-IP)实验检测Survivin蛋白乙酰化水平。取各组细胞,继续培养12、24、36、48 h时,采用CCK-8法检测细胞增殖能力(以OD值表示)。取各组细胞,采用细胞划痕实验检测迁移能力(以细胞迁移距离表示),采用Transwell小室实验检测侵袭能力(以穿膜细胞数表示),采用Western blotting法检测VEGF信号通路相关蛋白AKT1、MDM2、Bcl-2、Caspase3。结果si-P300组细胞Survivin蛋白乙酰化水平均低于si-P300-NC组、NC组(P均<0.05)。si-P300组细胞培养12、24、36、48 h时的OD值均低于si-P300-NC组、NC组(P均<0.05)。si-P300组细胞迁移距离和穿膜细胞数均低于si-P300-NC组、NC组(P均<0.05)。si-P300组细胞AKT1、MDM2、Bcl-2蛋白相对表达量均低于si-P300-NC组、NC组(P均<0.05),Caspase3蛋白相对表达量均高于si-P300-NC组、NC组(P均<0.05)。结论转染P300下调质粒抑制P300表达后,TE-1细胞乙酰化水平下降,细胞增殖、迁移、侵袭能力降低,其机制可能与调控VEGF信号通路有关。

高温胁迫下刺参H3K9ac特征及HATs、HDACs的表达

为了探索高温胁迫下刺参(Apostichopus japonicus)H3K9乙酰化水平的变化,研究组蛋白乙酰转移酶(Histone acetyltransferases,HATs)和去乙酰化酶(Histone deacetylases,HDACs)对刺参H3K9乙酰化的影响。本研究通过Western Blot技术研究高温胁迫条件下刺参肠道组织H3K9乙酰化特征,并对刺参基因组中4种组蛋白乙酰转移酶基因ajKAT2B、ajKAT5、ajKAT7、ajKAT8和4种组蛋白去乙酰化酶基因ajSIRT1、ajHDAC1、ajHDAC3、ajHDAC4进行结构解析和系统进化树分析;利用qRT-PCR定量研究在高温胁迫后刺参的8种基因表达量的变化模式。研究表明,通过Western Blot发现,在26℃胁迫下,H3K9ac水平在胁迫48 h后明显上升(P<0.05),在96 h后又迅速下降(P<0.05),表明其在高温胁迫下调控基因转录可能起到重要作用。基因结构解析发现HATs和HDACs含有保守的结构域,通过构建基因系统进化树发现其与物种进化树呈一致的趋势,也表现出功能上的高度保守。qRT-PCR结果表明:4种HATs基因在高温胁迫48 h后均有显著的上升(P<0.05),在胁迫96 h后,ajKAT2B和ajKAT8降低至对照组的表达水平,ajKAT5迅速下降至显著低于对照组水平,ajKAT7稍有降低但仍显著高于对照组。4种HDACs基因中,ajSIRT1在高温胁迫6 h后有明显的上升,在胁迫48 h后显著高于对照组(P<0.05),在胁迫96 h后降低至对照组的表达水平;ajHDAC1在高温胁迫6 h后有明显下降(P<0.05),在胁迫48 h后迅速上升至显著高于对照组水平(P<0.05),然后降低至对照组水平;ajHDAC3和ajHDAC4在高温胁迫6 h后均显著上升(P<0.05),在胁迫48 h后降至对照组水平并继续下降。研究结果表明,刺参H3K9ac乙酰化水平在高温胁迫下表现出明显动态变化,多种HATs和HDACs在mRNA水平上均有明显响应,这可能是导致H3K9ac变化的因素之一。

组蛋白乙酰转移酶Tip60降表达的肺腺癌细胞侵袭迁移能力变化及其机制

目的观察组蛋白乙酰转移酶Tip60降表达的肺腺癌细胞侵袭迁移能力变化并探讨其机制。方法肺腺癌细胞系A549分为Tip60降表达组、降表达阴性对照组及对照组,Tip60降表达组、降表达阴性对照组分别转染Tip60干扰siRNA及阴性对照siRNA,对照组不进行转染。采用Western blotting法检测细胞SETDB1蛋白,实时荧光定量PCR法检测细胞SETDB1 mRNA。采用CCK-8法观察细胞增殖能力,Transwell小室实验观察细胞侵袭能力,划痕修复实验观察细胞迁移能力。使用Cistrome DB数据库的UCSC Browser功能搜索发现肺腺癌细胞A549中SETDB1启动子区H3组蛋白第9位赖氨酸(H3K9)和第27位赖氨酸(H3K27)残基存在明显富集峰,采用染色质免疫沉淀检测细胞SETDB1基因启动子区H3组蛋白H3K9、H3K27的乙酰化水平。结果Tip60降表达组SETDB1蛋白及mRNA表达均小于对照组、降表达阴性对照组(P均<0.05)。培养24、48、72 h时,Tip60降表达组细胞增殖能力均小于对照组、降表达阴性对照组;Tip60降表达组侵袭细胞数少于对照组、降表达阴性对照组;培养24、48 h时Tip60降表达组划痕愈合率均小于对照组、降表达阴性对照组(P均<0.05)。Tip60降表达组SETDB1启动子区组蛋白H3K9及H3K27乙酰化水平均小于对照组、降表达阴性对照组(P均<0.05)。结论Tip60降表达可抑制肺腺癌细胞的侵袭迁移能力,其机制可能与调控SETDB1启动子区H3组蛋白乙酰化水平有关。

ATAT1 deficiency enhances microglia/macrophage-mediated erythrophagocytosis and hematoma absorption following intracerebral hemorrhage

MIcroglia/macrophage-mediated erythrophagocytosis plays a crucial role in hematoma clearance after intracerebral hemorrhage.Dynamic cytoskeletal changes accompany phagocytosis.However,whether and how these changes are associated with microglia/macrophage-mediated erythrophagocytosis remain unclear.In this study,we investigated the function of acetylatedα-tubulin,a stabilized microtubule form,in microglia/macrophage erythrophagocytosis after intracerebral hemorrhage both in vitro and in vivo.We first assessed the function of acetylatedα-tubulin in erythrophagocytosis using primary DiO GFP-labeled red blood cells co-cultured with the BV2 microglia or RAW264.7 macrophage cell lines.Acetylatedα-tubulin expression was significantly decreased in BV2 and RAW264.7 cells during erythrophagocytosis.Moreover,silencingα-tubulin acetyltransferase 1(ATAT1),a newly discoveredα-tubulin acetyltransferase,decreased Ac-α-tub levels and enhanced the erythrophagocytosis by BV2 and RAW264.7 cells.Consistent with these findings,in ATAT1-/-mice,we observed increased ionized calcium binding adapter molecule 1(Iba1)and Perls-positive microglia/macrophage phagocytes of red blood cells in peri-hematoma and reduced hematoma volume in mice with intracerebral hemorrhage.Additionally,knocking out ATAT1 alleviated neuronal apoptosis and pro-inflammatory cytokines and increased anti-inflammatory cytokines around the hematoma,ultimately improving neurological recovery of mice after intracerebral hemorrhage.These findings suggest that ATAT1 deficiency accelerates erythrophagocytosis by microglia/macrophages and hematoma absorption after intracerebral hemorrhage.These results provide novel insights into the mechanisms of hematoma clearance and suggest ATAT1 as a potential target for the treatment of intracerebral hemorrhage.