S_(180)肉瘤细胞对小鼠基因组DNA结构变化影响的核磁共振氢谱~1H—NMR研究

本实验利用核磁共振技术,对体内培养了腹水癌小鼠肝细胞基因组DNA的结构变化进行了测试,发现随致癌时间的延长,基因组DNA内胸腺嘧啶核苷酸周围的化学环境发生了定向的变化,造成DNA核磁共振氢谱中,部分胸腺嘧啶的甲基质子发生了化学位移,在′H-NMR图谱中1.9ppm峰Ⅰ减小,而2.6-2.7ppm区域的峰Ⅱ却明显增加,结果使双峰比(峰Ⅰ积分面积/峰Ⅱ积分面积)显著下降,本文为研究癌症发生和发展的遗传学机制提供了一个新的方法.     

核酸及其与组蛋白相互作用的~1H、~(13)C、~(31)P核磁共振研究

本文报道了酵母、大白鼠正常肝、大白鼠肝癌、大白鼠再生肝、大白鼠胚胎肝等的RNA的低场区质子主峰化学位移是各不相同的。酵母RNA的~(13)C谱能观察到24组碳原子峰,其~(31)P核磁共振信号出现在1.20ppm处;半高宽为40Hz。小牛胸腺、大白鼠肝和大白鼠脾等的组蛋白~1H谱的化学位移和波谱特征是相同的。小牛胸腺组蛋白的~(13)C谱可观察到5组碳原子峰。大白鼠脾组蛋白与酵母RNA相互作用后,组蛋白的质子峰变弱。酵母RNA与小牛胸腺组蛋白相互作用后,RNA低场Ⅱ区和Ⅰ区的质子峰变弱;~(31)P核磁共振信号完全消失。聚核苷酸I:C与组蛋白相互作用后,~(31)P核磁共振信号完全消失;聚核苷酸C与组蛋白相互作用后,~(31)P核磁共振信号强度约降低一半左右。     

杨尺蠖核型多角体病毒的DNA特性

本文报道了AciNPV-DNA的纯化及特性,证明纯化DNA具有典型紫外吸收光谱,在Sepharose 2B柱层析和超离心沉降分析中呈单一峰,Tm值为70.2℃,其(G+C)％=39.8％,增色效应为34％,限制性内切酶Pst Ⅰ,PvuⅡ,SaI Ⅰ,Eco R Ⅰ,BgI Ⅰ,XhoⅠ,Bgl Ⅱ酶切DNA,分别产生4,6,22,20,11,5,7个DNA片段,某些酶切电泳图谱中观察到亚分子片段,Bgl Ⅱ和EcoR Ⅰ双醇切DNA分子产生26个片段,Eco R Ⅰ酶切片段积加DNA分子量为56.55×10~6道尔顿,约85.70千碱基对,电镜观察DNA分子有线状和环状,环状分子长约27.38μ,分子量约为53.94×10~(?)道尔顿,以Eco R Ⅰ对1981～1985年林间连续复制回收的AciNPV的DNA同源性进行检测,结果无变化,AciNPV攻毒枣尺蠖幼虫所得枣尺蠖NPV,其DNA的Bgl Ⅱ图谱与AciNPV-DNA的Bgl Ⅰ酶谱有明显的差异。     

胃粘膜正常和癌变细胞基因组DNA的表面增强拉曼光谱研究

通过测试胃粘膜正常上皮细胞、高、中、低和未分化胃癌细胞基因组DNA的表面增强拉曼光谱,分析各自特征性谱峰。结果表明：正常和高分化细胞基因组DNA在1060cm^-1。被抑制,只在1010cm^-1。被激活,但后者的谱峰更强且出现“红移”,其DNA链结构出现不稳定;中、低、未分化细胞基因组DNA中以上两种振动模式都被激活,说明它们的DNA链出现了断裂,且呈现渐强的趋势。同时也说明了脱氧核糖基在恶性程度越高的细胞基因组DNA中带正电趋势越强。1100—1670cm^-1谱带属于dC,dG,dA,dT的综合振动叠加,恶性程度越高的癌细胞中更多的模式被激活。可见,分化越低的胃癌细胞基因组DNA具有更多的振动模式被激活,与正常的差异更明显,并且DNA链整体带正电的趋势也可能越强,提示可能有更多的氧化反应发生。     

鸡卵黄DNA的存在、提取和特性分析

利用免疫细胞化学技术证实了鸡蛋卵黄球中DNA的存在 .利用Ficoll 40 0密度梯度离心纯化卵黄球并提取了DNA .限制性内切酶分析表明所得卵黄DNA是序列不均一的DNA分子群 .MspⅠ和HpaⅡ分析显示该DNA的甲基化程度极低 .Southern杂交表明卵黄DNA含有基因组DNA的一小部分序列 .并认为卵黄DNA是一种独特的有可能在鸡胚早期发育中发挥功能的DNA .     

疫苗

960999 禽痘病毒DNA复制及作图分析[英]/Zantinge, J. L.…//Can. J. Microbiol. -1995,41(4～5).-378～387[译自DBA,1995,14(17),95-10127] 产生了一种禽痘病毒Chick-N-Pox疫苗株DNA基因组的EcoRⅠ、BamHⅠ、NcoⅠ和PstⅠ限制性物理图谱。利用琼脂糖凝胶电泳和PAGE分析了EcoRⅠ、BamHⅠ、BglⅡ、NcoⅠ、StyⅠ、PstⅠ和PvuⅡ消化DNA的凝胶分布结果。从中估测禽痘病毒基因组的     

胰岛素与类似物结构与功能关系的核磁共振研究

对胰岛素及其类似物DPI(去五肽胰岛素),DOI(去八肽胰岛素)的~1H NMR(核磁共振)变温谱进行了比较研究。胰岛素的三维结构随温度上升发生明显的变化,以Val、Leu、Ile的CH_3组成的高场峰区(0.2～1.1ppm)的峰形逐渐变尖变窄,特别突出的是0.34～0.42ppm峰肩当温度升高至50℃以上逐渐向低场移动以至消失。此峰肩为A_2-Ile的两个CH_3受邻近A_(19)-Tyr芳香环的环流场屏蔽影响的高场位移峰。DPI变温谱具有与胰岛素十分相似的变化。而对比DOI的变温谱,我们发现常温时,由于A链头尾紧密联系的极性区构象已遭破坏,因而不出现此峰肩。此局部构象可能与DPI保存有相当大的生物活性和DOI丧失生物活性有关。胰岛素的A链头尾三维构象随温度上升至50℃以上的明显变化与胰岛素和受体结合活性在温度高于50℃被破坏的结果是一致的。这就提示了结合活性的变化与某些三维构象的变化可能是相关的。这也支持了A链头尾形成的极性区很象是胰岛素“结合部位”的一部分。     

化学诱导的B95-8细胞中TK酶的分离和性质研究

巴豆油和正丁酸钠(nSB)诱导Raji和B95-8细胞株生成胸腺嘧啶核苷激酶(TK),其粗提液,经DEAE—纤维素柱层析,可分成两个性质不同的TK活性峰—峰Ⅰ和峰Ⅱ:(1)峰Ⅰ是穿过峰,峰Ⅱ为洗脱峰,在120mMol/L从K_2HPO_4缓冲液时洗脱下来;(2)峰Ⅱ含量在病毒生产性细胞B95-8中高于非生产性的Raji细胞;(3)B95-8细胞经联合诱导48小时后,峰Ⅱ比活性最高;(4)TTP对峰Ⅰ和峰Ⅱ的抑制效应不同,两峰利用GTP能力也不同;(5)PAGE结果表明:峰Ⅰ的Rm值为0.044,峰Ⅱ呈现两条带,Rm值分别为0.015和0.276;(6)峰Ⅰ的Km值为0.86μMol/L,峰ⅡKm值为0.29μMol/L。根据以上的结果,我们认为:峰Ⅰ是细胞TK(C-TK),而峰Ⅱ具有许多疱疹病毒TK的特性,因此,峰Ⅱ是EB病毒相关TK(EBV-TK)。     

小麦与高冰草体细胞杂种后代的核基因组和叶绿体基因组的遗传特征

以小麦(Triticum aestivum L.)与高冰草(Agropyron elongatum(Host)Nevski)体细胞杂种同一个克隆来源的F2-F6自交系Ⅱ-2、Ⅱ-Ⅰ-8以及由Ⅱ-Ⅰ-8 F2分离形成的8-1(F3-F6)为材料,利用小麦叶绿体基因组的微卫星(Microsatellite)特异引物及随机扩增多态性DNA(RAPD)引物进行分析.结果表明,杂种株系的叶绿体基因组组成一致,均以小麦叶绿体基因组为主,仅在rpl14和rpl16基因的间隔序列中检测到双亲的特征带,表明有高冰草的叶绿体DNA在杂种中存在,并稳定遗传至第六代.RAPD分析表明,不同杂种株系中存在不同的高冰草核DNA片段,核基因组在传代中基本稳定.     

小麦与高冰草体细胞杂种后代的核基因组和叶绿体基因组的遗传特征

以小麦(TriticumaestivumL.)与高冰草(Agropyronelongatum(Host)Nevski)体细胞杂种同一个克隆来源的F2-F6自交系Ⅱ-2、Ⅱ-Ⅰ-8以及由Ⅱ-Ⅰ-8F2分离形成的8-1(F3-F6)为材料,利用小麦叶绿体基因组的微卫星(Microsatellite)特异引物及随机扩增多态性DNA(RAPD)引物进行分析。结果表明,杂种株系的叶绿体基因组组成一致,均以小麦叶绿体基因组为主,仅在rpl14和rpl16基因的间隔序列中检测到双亲的特征带,表明有高冰草的叶绿体DNA在杂种中存在,并稳定遗传至第六代。RAPD分析表明,不同杂种株系中存在不同的高冰草核DNA片段,核基因组在传代中基本稳定。     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

Demonstration of a macrophage migration enhancement factor in the sera of young rabbits

Alveolar macrophages (AM), harvested from the lungs of untreated normal young rabbits (New Zealand White) 14 days to 8 weeks of age, exhibited a state of migration stimulation compared to AM from normal adult rabbits (5 to 6 months of age). Migration of AM from normal adult rabbits (New Zealand White) was stimulated 2.0- to 2.5-fold when incubated with sera from 39- to 46-day-old rabbits compared with sera from normal adult rabbits. Furthermore, 4-day spleen cultures obtained from animals 28 to 59 days of age yielded supernatants that also stimulated the migration of adult AM. The spleen cell culture supernatants from 42- to 49-day-old animals had the greatest activity and stimulated the migration of adult AM 2.5- to 3.2-fold compared to the supernatants from adult normal rabbits. The peak production of migration enhancement factor (MEF) by splenic lymphoid cells coincided with the peak activities found in the sera. It was observed that nonadherent peanut agglutinable lymphoid cells produced MEF. When sera or culture supernatants containing MEF were mixed with MIF-containing adult sera or spleen cell culture supernatants, the respective activities were neutralized. The large migrations of normal neonatal AM were diminished by the addition of MIF-containing sera obtained from BCG-sensitized/challenged rabbits. In contrast, AM from BCG-sensitized rabbits, which exhibited a state of reduced migration, were enhanced by MEF-containing sera from untreated young rabbits. Three peaks of MEF activity were detected in Sephadex G-100 column fractionated sera from 42-day-old rabbits having MWs of approximately (Peak I) 80,000, (Peak II) 43,000, and (Peak III) 8000 to 18,000; most of the activity was found in peaks II and III. Two peaks of MEF activity were detected in Sephadex G-100 column-fractionated spleen cell culture supernatants from 42-day-old rabbits having MWs of approximately (Peak I) 35,000 to 43,000 and (Peak II) 10,000 to 14,000; most of the activity was in peak I which corresponds to peak II of the serum fractionation experiment. Collectively, these data indicate that MEF is a lymphokine that could be important in the modulation of cell-mediated immune effector responses.     

关于链格孢的毒性及其产毒条件的研究

本文报告了从食管癌高发区林县分离的具有强烈毒性和致突变性的链格孢(Alternariaalternata)“D_(10)-A_2”菌株粗提物半数致死量的测定,结果为71.25g 培养物/kg 体重;7个菌株粗提物的程序外 DNA 合成(UDS)试验,6个菌株粗提物的 DNA 合成抑制(DSI)试验。结果 UDS 试验6个菌株为阳性,DSI 试验5个菌株为阳性。说明互隔交链孢提取物可致人体细胞 DNA 的损伤。本文还报道了链格孢适宜的产毒条件。链格孢在一般粮食上均能生长繁殖产毒,适温为25℃,适宜的湿度根据粮食品种不同而为33—50％含水量。在北方侵染的主要粮食为小麦,当麦粒灌浆成熟期遇到较长时间的阴雨天气,或在收割脱粒时遇到连阴雨,麦粒被雨水浸泡。污染非发生于贮存期。     

Chromatographic resolution of two DNA polymerase activities from bloodstream forms of Trypanosomabrucei: Differential responses to exogenous polyamine addition

A single peak of DNA polymerase activity from extracts of $\text{T.}\text{brucei}$, obtained by DEAE-cellulose and phosphocellulose ion-exchange chromatography, was resolved into two peaks differing in KCl concentration necessary to elute them from a DNA-agarose column. Peak I (eluting at 0.2 M KCl) and Peak II (eluting at 0.4 M KCl), differed in response to increasing KCl concentrations, although both functioned optimally with Mg²⁺ as divalent cation when DNA synthesis was directed either by activated DNA or poly (dC)·(dG)_12–18. Due to the potential significance of polyamines in the metabolism of $\text{T.}\text{brucei}$, the effect of exogenous polyamine on rates of DNA synthesis by the peak I and II enzymes was compared with that of murine DNA polymerase alpha. Only the peak I enzyme was significantly stimulated (up to 4-fold) by the biologically active polyamines spermine and spermidine at physiological concentrations. The response of the peak I enzyme resembled that of the alpha polymerase. This result suggests a possible functional difference between peak I and II enzymes, as well as a potential target site for trypanocidal drug development.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.     

ONTOGENETIC DEVELOPMENT OF A PHOSPHODIESTERASE ACTIVATOR AND THE MULTIPLE FORMS OF CYCLIC AMP PHOSPHODIESTERASE OF RAT BRAIN

Abstract— The activity of cyclic AMP phosphodiesterase of rat cerebral homogenates increased several-fold between 1 and 60 days of age. Enzyme activity in the cerebellum, on the other hand, did not increase during this period. A kinetic analysis of the phosphodiesterase activity revealed evidence for multiple forms of the enzyme and indicated that the postnatal increase in phosphodiesterase activity of rat cerebrum was due almost exclusively to the high K_m enzyme. In cerebellum, the ratio of the high and low K_m enzyme remained fairly constant during ontogenetic development. Physical separation of the phosphodiesterases contained in 100,000 g soluble supernatant fractions of sonicated brain homogenates by polyacrylamide disc gel electrophoresis confirmed the presence of multiple enzyme forms. In adult rats we found six distinct peaks of phosphodiesterase activity (designated I to VI according to the order in which they were eluted from the column) in cerebellum and 4 forms of the enzyme (Peaks I through IV) in cerebrum. Brains of newborn rats had a different pattern and ratio of phosphodiesterase activities. For example, Peak I phosphodiesterase was undetectable in cerebrum or cerebellum of newborn rats. Moreover, in the cerebellum of newborn rats Peak II was the dominant peak whereas in the cerebellum of adult rats Peak III was the largest peak. A comparison of the multiple forms of phosphodiesterase from the cerebrum of newborn and adult animals suggested that the postnatal increase in phosphodiesterase activity previously seen in crude homogenates was due largely to an increase in a high K, Peak II phosphodiesterase. The ratios of activities of the other peaks and their sensitivities to an activator of phosphodiesterase were similar in newborn and adult rats. An endogenous heat-stable activator of phosphodiesterase was found in cerebrum, cerebellum and brain stem. In newborn rats, the cerebellum contained several-fold less activity of this activator than did cerebrum or brain stem. However, the activity of this activator increased with age in the cerebellum and would appear to have decreased postnatally in cerebrum and brain stem. These results suggest that some multiple forms of phosphodiesterase can develop independently and that changes in activities of these phosphodiesterases may occur by increases in the quantity of enzyme or by changes in the quantity of an endogenous activator of phosphodiesterase.     

Epinephrine effects on cyclic AMP-dependent protein kinases from rat diaphragms

Diaphragm extracts were subjected to electrophoresis on polyacrylamide gels to separate the different molecular species of th cyclic AMP-dependent protein kinase. Using cyclic [3H]AMP, three peaks of binding activity were observed. The peak closest to the origin (peak I) was associated with cyclic AMP-dependent protein kinase activity and was abolished by incubation of the extracts with cyclic AMP prior to electrophoresis. The peak farthest from the origin (peak III) was devoid of kinase activity and was increased by incubation of extracts with cyclic AMP before electrophoresis; furthermore, when extracts were incubated with cyclic [3H]AMP before electrophoresis, essentially all the radioactivity appeared in peak III. Peak II, in an intermediate position, was also abolished by preincubation of the extracts with cyclic AMP and both its binding capacity and cyclic AMP-dependent protein kinase activity were lower than in Peak I. A peak of cyclic AMP-independent protein kinase (peak 0) that migrated more slowly than peak II was also detected. From these and other data it is concluded that peaks I and II are cyclic AMP-dependent protein kinase and that peak III is the dissociated regulatory subunit, respectively. Peak 0 is cyclic AMP-independent protein kinase together with free catalytic subunits from cyclic AMP-dependent protein kinase. Incubation of rat diaphragms with epinephrine resulted in dose- and time-dependent decrease in peak I and increase in peak III. These changes correlated with the decrease of cyclic AMP-dependent protein kinase associated with peak I. No changes in Peak II were observed with epinephrine, but an increased peak 0 was noted. Changes in peak I and peak III correlated with the modification of glycogen synthase and glycogen phosphorylase activities. No regulatory subunits (peak III) were detected as phosphorylated forms in diaphragms previously equilibrated with 32P. Treatment with epinephrine produce no noticeable phosphorylation of these regulatory subunits.     

Separation and characterization of two carnosine-splitting cytosolic dipeptidases from hog kidney (carnosinase and non-specific dipeptidase)

High performance anion-exchange chromatography was used to separate two carnosine-hydrolysing dipeptidases from hog kidney. Both enzymes (peaks I and II) were cytosolic and were activated and stabilized by Mn2+ and dithiothreitol. Peak I had a narrow specificity when assayed without added metal ions, but a broad specificity in the presence of Mn2+ or Co2+. Peak II was inactive unless both Mn2+ and dithiothreitol were present. Bestatin and leucine inhibited peak II, but not peak I. Peak I had a Km of 0.4 mM carnosine, a pI of 5.5 and a Mr of 57,000. Peak II had a Km of 5 mM carnosine, a pI of 5.0 and a Mr of 70,000. Hog and rat brain and liver carnosinase activity was completely inhibited by bestatin, indicating that these organs contained peak II, with little or no peak I enzyme. Hog kidney peak I contained the classical carnosinase of Hanson and Smith, who first described this enzyme. It also contained activity against homocarnosine ("homocarnosinase") and showed "manganese-independent carnosinase" activity. These three activities could not be separated using 8 different chromatographic procedures; it was concluded that they are attributable to one enzyme. It is recommended that the name carnosinase be retained for this enzyme and the names "homocarnosinase" and "manganese-independent carnosinase" be withdrawn. The properties of hog kidney peak II closely resembled those of human tissue carnosinase (also known as prolinase, a non-specific dipeptidase), mouse "manganese-dependent carnosinase" and a rat brain enzyme termed "beta-Ala-Arg hydrolase". Since these terms appear to represent closely related enzymes with broad specificity, the recommended name for each is "non-specific cytosolic dipeptidase".     

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.