~(60)Co-γ辐照对中国仓鼠卵巢成纤维细胞DNA和组蛋白合成的影响

本文介绍了~(60)Co-γ辐照对同步的和非同步的CHO细胞的DNA合成和组蛋白合成关系的影响的研究,用~3H-胸腺嘧啶核苷和~(14)C-丙氨酸双标记,未经辐照的和经4Gy～60Gy ~(60)Co-γ辐照的CHO细胞,通过~3H和~(14)C的参入来估价DNA和组蛋白的合成,并用聚丙烯酰胺凝胶电泳鉴定辐照前后组蛋白各组分的变化情况,实验表明: 1)、在4～60Gy剂量范内,无论是同步的还是非同步的CHO细胞其DNA合成和组蛋白合成都受到不同程度的抑制。2)、在辐照后1—3小时,DNA合成和组蛋白合成都受到不同程度的抑制,但辐照后4小时,DNA合成被进一步抑制而组蛋白的合成却逐渐恢复正常,到辐照后48小时组蛋白的合成几乎接近对照水平。3)、16Gy ~(60)Co-γ辐照后8小时,非同步的CHO细胞的DNA合成被抑制的情况比G_1期CHO细胞更为严重。4)、16Gy ~(60)Co-γ辐照S期细胞,在辐照后1—24小时中DNA合成被明显抑制的同时,组蛋白的合成也受到相应的抑制。5)、从未经辐照的和经6、16和60Gy~(60)Co-γ辐照的CHO细胞分别提取全组蛋白,进行聚丙烯酰胺凝胶电泳,从电泳图谱的变化清楚地看到组蛋白H_1和H_3受辐照影响大于组蛋白H_4和H_(2B)+H_(2A),因此我们推测DNA合成和组蛋白H_1和H_3的关系较之组蛋白H_4和H_(2A)+H_(2B)更为密切。     

砂鼠利什曼原虫(Leishmania gerbilli)染色质与动基体碱性蛋白的初步研究

利用纯化的砂鼠利什曼原虫细胞核作为起始材料对其染色质碱性蛋白进行分析,发现这类生物中只存在四种核芯组蛋白(H_4,H_2A,H_2B和H_3)。 用凝胶电泳比较全细胞的与细胞核的碱性蛋白时,检出了一种来自细胞质的酸溶性蛋白(L组分)。细胞化学的检测表明它定位于动基体(Kinetoplast)。     

对黑斑蛙(Rana nigromaculata)和中华大蟾蜍(Bufo bufo asiatius)精子染色质的碱性蛋白和超微结构的比较研究

在精子形成过程中,精子细胞的细胞核高度浓缩成结构致密,体积很小的细胞核(精子核)。为了研究这种极度浓缩之染色质的组分和结构,我们用凝胶电泳法分析了黑斑蛙和蟾蜍的经过提纯之精子的染色质碱性蛋白和用电镜铺片法观察这些精子染色质的亚显微结构。我们发现黑斑蛙精子含有五种组蛋白,即H_1、H_3、H_(2B)、H_(2A)和H_4。H_3、H_(2A)、H_(2B)和H_4是核小体的主要组分。用电镜观察黑斑蛙精子染色质的结果说明,黑斑蛙精子染色质含有核小体结构,反之电泳分析结果说明,蟾蜍精子含有4条碱性蛋白带,其中1条带染色很深、宽度很大,迁移率远较组蛋白为大而与鱼精蛋白相似,而另外3条带染色既浅、宽度又狭,其中有1条从其迁移率来看相当于H_(2B)。用电镜观察时,这种蟾蜍精子染色质没有核小体结构。     

生肌素引起组蛋白H3和H4高度乙酰化及染色质溶解性增加

为研究生肌素在染色质重建过程中的作用 ,采用生肌素真核表达载体转染C2C12肌细胞 ,细胞核经微球菌核酸酶消化后 ,提取DNA进行SDS PAGE分析 .生肌素转染的细胞 ,其核小体 (染色质 )在Mg2 + 溶液中的溶解性明显增加 ,提示染色质组蛋白乙酰化程度提高 .组蛋白经TAU SDS(2 D)双相凝胶电泳分析 ,发现在转染生肌素真核表达载体 2 4h后 ,组蛋白H4的乙酰化修饰程度最高 .采用抗乙酰化组蛋白H3和H4抗体进行的Western印迹分析进一步证明了乙酰化的发生 .上述变化与染色质的活跃程度相关 .RT PCR结果显示 ,生肌素的靶基因烟碱样乙酰胆碱受体 (nAChR)α亚基和肌酸激酶 (MCK)基因在转染后表达水平提高 .结果提示 ,强制性表达外源性生肌素可引起肌细胞核染色质重建 ,进而激活靶基因     

与大鼠Morris肝癌7777相关的染色质蛋白的免疫亲和层析分离与鉴定

 用5mol/L尿素,将大鼠Morris肝癌7777染色质解离为染色质非组蛋白 (UP组分)及染色质沉淀(UC组分)。UP(含90—95％非组蛋白)用免疫亲和层析(与大鼠Morris肝癌7777去组蛋白染色质抗体交联)分级,经2mol/L NaSCN及8mol/L尿素分部洗脱。将UP及UC,来自UP亲和层析的2mol/L NaSCN及8mol/L尿素洗脱组分同时进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)。以大鼠Morris肝癌7777去组蛋白染色质抗体作探针,进行免疫显迹(Immunoblot)测定。在UP部分出现二条阳性带,分子量为:200K及116K。UC部分有三条染色不很深的阳性带,分子量为200K,118K及91K。来自UP亲和层析的2mol/L NaSCN及8mol/L尿素洗脱部分分别有一条浓而清晰的阳性带,分子量分别为74K及83K。用酶联免疫吸附法(E1isa)测试从UP凝胶上切割下的阳性区带,其免疫特异性显著。     

增强Na+-Ca2+交换对大鼠心脏的正性变力作用及对哇巴因效应的加强

本文采用大鼠乳头肌张力测定及离体心脏灌流技术,研究大鼠心肌Na -Ca2 交换对乳头肌及离体灌流心肌变力性的影响。采用大鼠特异性Na -Ca2 交换激动剂E-4031能剂量依赖性地增加大鼠乳头肌的发展张力(P<0.05,n=6)及离体心脏的心泵功能(P<0.05,n=4);特异性Na -Ca2 交换抑制剂KB-R7943具有相反的效应,并可完全消除E-4031引起的正性变力作用。哇巴因(ouabain,0.5μmol/L)与E-4031(3μmol/L)联合使用,可使乳头肌发展张力由单独使用哇巴因时的0.25±0.03 g升高至0.29±0.04g(P<0.05,n=6);联合用药对大鼠离体心脏心泵功能的影响也强于哇巴因单独作用的效果。本研究结果证实,E-4031通过增强心肌Na -Ca2 交换,对大鼠乳头肌和离体心脏产生正性变力作用;与哇巴因合用时,它们的正性变力作用有相加作用。     

组蛋白去乙酰化酶抑制剂对大鼠应激性心肌损伤的保护作用

目的:观察组蛋白去乙酰化酶抑制剂在应激性心肌损伤发生过程中的作用。方法:健康雄性Wistar大鼠随机分为3组(n=6),用束缚应激方法建立慢性应激性心肌损伤模型,采用组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)干预,观察TSA对应激性心肌损伤的保护作用。Western blot检测实验各组大鼠心肌的组蛋白乙酰化水平,采用分光光度法动态监测大鼠血清乳酸脱氢酶(LDH)和肌酸激酶同工酶-MB(CK-MB)活性以及心肌组织Caspase 3活性,Nagar Olsen染色观察心肌的早期损伤。结果:束缚应激可以显著降低大鼠心肌的组蛋白乙酰化水平(P0.05),而TSA干预可以抑制应激所致的心肌组蛋白乙酰化水平降低(P0.05);束缚应激可以引起大鼠血清LDH和CK-MB活性、心肌组织Caspase 3活性显著升高(P0.05),发生心肌早期损伤,而TSA干预可显著降低束缚应激引起的LDH(P0.05)、CK-MB活性(P0.05)、Caspase 3活性升高(P0.05)。结论:组蛋白去乙酰化酶抑制剂TSA对应激性心肌损伤具有一定的保护作用。     

亚精胺诱导自噬的作用机制

亚精胺(spermidine)是含有3个胺基的低分子量脂肪族碳化物,是存在于所有生物体中的天然多胺之一。自噬(autophagy)对于降解细胞内受损蛋白质和细胞器是必需的。外源性亚精胺可作为自噬的天然诱导剂,并且是安全和无毒的。新近研究表明,亚精胺可通过AKT/AMPK-FoxO3-Atg途径诱导自噬,还能促进组蛋白脱乙酰基酶4(histone deacetylase 4,HDAC4)向细胞核转运,降低细胞质HDAC4含量,进而增强微管相关蛋白1S(microtubule-associated protein 1S,MAP1S)乙酰化和稳定性以激活自噬。此外,亚精胺可作为乙酰转移酶抑制剂调节EP300活性,进而改变Atg5、Atg7、LC3和Atg12的乙酰化状态。同时,还可通过诱导哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)去磷酸化,激活ULK1/2-Atg13-FIP200复合物参与调控动物机体内的自噬过程。本文就自噬概念和亚精胺诱导自噬作用途径的最新研究进展作一综述。     

雌二醇受体在兔子宫内膜染色质蛋白中的分布

本文利用不连续HAP层析法解离兔子宫内膜染色质蛋白,共分离出四个主要组分:(1)疏松结合的非组蛋白组分(0.25M NaCl解离的),(2)组蛋白组分(2M NaCl解离的),(3)紧密结合的非组蛋白组分(2M NaCl/5M尿素解离的)和(4)很紧密结合的非组蛋白组分(5M尿素/4M盐酸胍解离的)。在离体条件下使部分纯化的雌二醇受体复合物(RE)与兔子宫内膜染色质结合,然后进行羟磷灰石层析,用四种溶剂解离四种染色质蛋白组分,此时RE也随同被解离下来,后者与染色质蛋白第1、2、3和4组分结合的相对结合量分别为0.33±0.10,0.10±0.03,0.32±0.03和0.21±0.05,表明RE主要与非组蛋白组分结合,占86%。当己烯雌酚受体复合物存在时,RE与染色质蛋白的结合受到抑制。又初步比较了雌三醇和雌二醇受体复合物与染色质蛋白的结合量,观察到雌三醇受体复合物的结合量小得多,这种现象符合于雌三醇作为一种较弱的雌激素的特性。     

多胺及其在植物体内的生理作用

多胺研究的历史及其在植物体内的分布多胺(polyamines)是一类广泛存在于原核生物及真核生物中的生物学活性物质,是一类低分子脂肪族含氮碱。常见的多胺(包括二胺)有腐胺(putrescine,put)H_2N(CH_2)_4NH_2、尸胺(cadaverine,cad)H_2N(CH_2)_5NH_2、亚精胺(spermidine,spd)H_2N(CH_2)_4NH(CH_2)_3NH_2和精胺(spermine,spm)H_2N(CH_2)_4NH(CH_2)_4NH(CH_2)_3NH_2。亚精胺又称精脒,是     

Isolation, identification, and characterization of histones from plasmodia of the true slime mold Physarum polycephalum using extraction with guanidine hydrochloride

Histones from plasmodia of the true slime mold Physarum polycephalum have been prepared free of slime by an approach to histone isolation that uses extraction of nuclei with 40% guanidine hydrochloride and chromatography of the extract on Bio-Rex 70. This procedure followed by chromatography or electrophoresis has been used to obtain pure fractions of histones from Physarum microplasmodia. Physarum microplasmodia have five major histone fractions, and we show by amino acid analysis, apparent molecular weight on three gel systems containing sodium dodecyl sulfate, mobility on gels containing Triton X-100, and other characterizations that these fractions are analogous to mammalian histones H1, H2A, H2B, H3, and H4. Significant differences between Physarum and mammalian histones are noted, with histone H1 showing by far the greatest variation. Histones H1 and H4 from Physarum microplasmodia have similar, but not identical, products of partial chymotryptic digestion compared with those of calf thymus histones H1 and H4. Labeling experiments, in vivo, showed that histone H1 is the major phosphorylated histone and approximately 15 separate phosphopeptides are present in a tryptic digest of Physarum histone H1. The core histones from Physarum, histones H2A, H2B, H3, and H4, are rapidly acetylated; histone H4 shows five subfractions, analogous to the five subfractions of mammalian histone H4 (containing zero to four acetyllysine residues per molecule); histone H3 has a more complex pattern that we interpret as zero to four acetyllysine residues on each of two sequence variants of histone H3; histones H2A and H2B show less heterogeneity. Overall, the data show that Physarum microplasmodia have a set of histones that is closely analogous to mammalian histones.     

Butyrate-induced histone hyperacetylation in human and mouse cells: estimation of putative sites of histone acetylation in vivo

Human and mouse cells in culture were treated with various concentrations of sodium butyrate. Acid-extracted histones of control and butyrate-treated cells were analyzed by two-dimensional gel electrophoresis. All core histones of the control cells contained modified forms. All core histones of the butyrate-treated cells were hyperacetylated. Depending on the number of acetylation sites per molecule, each histone or histone variant exhibited a characteristic number of acetylated forms. This number was the same for each histone common in human and mouse cells treated with butyrate. Histones 2A.1, 2A.2, and 2A.X have two sites of inner acetylation; 2A.Z has 3; 2B's have 5; and each one of the H3 variants as well as H4 have 4.     

Reversible effects of Na-butyrate on histone acetylation

Histones were labeled by incubating HeLa cells in the presence of radioactive leucine for 20 hours. Following a 5 hour chase in non-radioactive medium the cells were exposed to 7 mM Na-butyrate to increase the level of histone acetylation. Histones were then extracted, fractionated by high-resolution electrophoresis in acetic acid-urea gels and the specific activity of the parental form of H4 histone and that of each acetylated form was calculated. No differences were found in the specific activities indicating that the major effect of butyrate on histone acetyl levels involves histones which were synthesized before the administration of butyrate. The effect is reversible and within 15 minutes after the removal of the drug most of the acetylated forms of H4 histone are converted to the unmodified form.     

Histone variants and histone modifications in chromatin fractions from heterochromatin-rich Peromyscus cells

In order to investigate the relationship between condensed heterochromatin and histone modification by acetylation, phosphorylation and amino acid variation, chromatin from cultured Peromyscus eremicus cells, containing 35% constitutive heterochromatin, was fractionated into heterochromatin-enriched and heterochromatin-depleted fractions. The constitutive heterochromatin content of these fractions was determined from satellite DNA content. The distribution of phosphorylated and acetylated histones and amino acid variants of histone H2A in these chromatin fractions was examined by gel electrophoresis. Fractionation of histones demonstrated that endogenous histone phosphatase activity was high in chromatin fractions and could not be inhibited sufficiently to allow accurate histone phosphorylation measurements. However, sodium butyrate did inhibit deacetylation activity in the fractions, allowing histone acetylation measurements to be made. It was found that the constitutive heterochromatin content of these fractions was proportional to both their unacetylated H4 content and their more-hydrophobic H2A content. These observations support, by direct measurement, earlier experiments (Exp cell res 111 (1978) 373; 125 (1980) 377; 132 (1981) 201) suggesting that constitutive heterochromatin is enriched in unacetylated arginine-rich histones, and in the more hydrophobic variant of histone H2A.     

THE HISTONES ASSOCIATED WITH CONDENSED AND EXTENDED CHROMATIN OF MOUSE LIVER

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin. Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels. Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin. However, minor differences were found. A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2A1 and F2A2 differed in the two types of chromatin. The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples. Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions.     

Ultraviolet light induced preferential cross-linking of histone H3 to deoxyribonucleic acid in chromatin and nuclei of chicken erythrocytes

Histones have been cross-linked to DNA in chicken erythrocyte nuclei and chromatin by using ultraviolet light irradiation at 254 nm. Following irradiation, cross-linked histone-DNA adducts were isolated and purified by hydroxylapatite chromatography, and the DNA component was subjected to acid hydrolysis. Of several hydrolysis techniques investigated, trichloroacetic hydrolysis of the DNA component of the adducts was found to be most effective. Histones isolated from hydrolyzed histone-DNA adducts were characterized by gel electrophoresis and fingerprint analysis. No histone-histone protein adducts were observed. All histone fractions have been shown to cross-link DNA in nuclei or chromatin by utilizing the technique employed, but with different propensities. The order of observed cross-linking, deduced from kinetic experiments, is H1 + H5, H3 greater than H4 greater than H2A much greater than H2B. The preferential binding of the core histone H3, as compared to the other core histones, is discussed in light of recent data concerning histone-DNA interactions and nucleosome structure. The use of the ultraviolet light technique as a conformational probe to study chromatin is also discussed.     

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.     

Segregation of rapidly acetylated histones into a chromatin fraction released from intact nuclei by the action of micrococcal nuclease.

It has been previously shown that micrococcal nuclease digestion and subsequent fractionation of hen oviduct nuclei generates fractions enriched (first supernatant fraction - 1SF) and depleted (second supernatant fraction - 2SF) in ovalbumin genes, while a third fraction, the pellet fraction, contains about the same level of this gene as whole chromatin (Bloom and Anderson (1978) Cell 15, 141-150). We have utilized this fractionation method in an attempt to assess the extent and kinetics of histone acetylation associated with chromatin from the 1SF, 2SF, and pellet fraction. Hepatoma Tissue Culture (HTC) cells were labelled for 30 minutes in vivo with 3H-acetate, nuclei isolated and the chromatin fractionated. The specific activity of the histones in the 1SF was slightly greater than that of the 2SF (1.2 to 1.6 fold difference) independent of the length of nuclease digestion. If the labelling period is followed by short (10 to 60 minute) treatment of the cells with sodium butyrate, the more rapidly as well as more extensively acetylated histones are also preferentially found in the 1SF. This is in part the result of segregation of chromatin particles into the 1SF as the histones associated with these particles become hyperacetylated. That is, the extent of histone acetylation regulates the distribution of chromatin in the 1SF, 2SF and pellet fraction.