免疫荧光染色揭示草履虫接合生殖过程中静止原核的空间位置(英文)

在尾草履虫的接合生殖过程中,共有三次配前核分裂。在配前第三次分裂结束后,两个接合的细胞内均形成一个迁移原核和一个静止原核。迁移原核位于口旁锥内,而且紧贴于接合面,静止原核则位于迁移原核的外侧,两者呈左右排列,距离接近。但是,目前对导致两种原核近距离的原因尚不清楚。该文通过α-微管蛋白的单克隆抗体对受精核形成前的接合对进行了免疫荧光染色,结果发现,配前第三次分裂不同于前两次分裂,连接迁移原核和静止原核的核间连丝伸向细胞的后方,呈"U"或"V"型,结果导致两个原核左右排列,而不是前后排列,两者间的距离缩短。这个结果也阐明了造成两种原核近距离的原因。     

不同染色方法揭示纤毛原生动物草履虫接合生殖过程的新细节

通过石炭酸品红、Hoechst 33342、蛋白银及免疫荧光标记等染色方法对草履虫接合生殖过程进行了重新观察,结果发现:1)新月核是第一次减数分裂前期小核的主要形态学特征,在核内有一未被石炭酸品红、Hoechst 33342着色区域,蛋白银染色则清楚显示该结构;2)4个单倍体减数分裂产物中的1个核进入口旁锥完成配前第三次核分裂,其余3核退化.蛋白银染色和抗α微管蛋白单克隆抗体进行免疫荧光标记显示,核进入口旁锥的时期在第二次减数分裂末期而非减数分裂结束后;3)配前第三次分裂末期,核间连丝的中间段有一被蛋白银识别的结构,但免疫荧光标记却无显示,只表现为纤维状结构与两侧核间连丝相连.观察结果为草履虫接合生殖过程中相关分子生物学机制研究奠定了必要的形态学基础.     

韭菜的小孢子四分体微核和核型的初步研究

本文研究了昆明栽培的韭菜的小孢子染色体数目和核型,多数小孢子的染色体数目ｎ＝１６,少数是非整倍体,核型公式为ｎ＝２ｘ＝１６＝１４ｍ＋２ｓｔ（ＳＡＴ）。同时观察了韭菜的育性和小孢子四分体微核情况,育性和四分体微核率分别为４８％、２３６％。上述结果说明韭菜的核型与前人曾报道过的韭菜的核型一样是同源四倍体。     

韭菜的小孢子四分体微核和核型的初步研究

本文研究了昆明栽培的韭菜的小孢子染色体数目和核型,多数小孢子的染色体数目n=16,少数是非整倍体,核型公式为n=2x=16=14m+2st(SAT)。同时观察了韭菜的育性和小孢子四分体微核情况,育性和四分体微核率分别为48％、23.6％。上述结果说明韭菜的核型与前人曾报道过的韭菜的核型—样是同源四倍体。     

猫小脑第Ⅶ小叶皮层—核团投射的电生理学研究

在三碘季铵酚制动的去大脑猫上,记录了小脑后叶的第Ⅶ小叶皮层浦肯野细胞(PC)对分别刺激顶核、间位核和齿状核的逆行场电位和逆行单位反应,以确定小脑皮层PC对这三个核团投射的空间分布。在鉴定了PC对其靶核团的投射后,用特制的模拟自然屈腕运动的刺激装置来推动猫同侧前肢的掌背,造成腕关节一次轻微的屈曲,观察该PG对这一刺激的单位反应。实验资料用电子计算机处理,作出平均诱发电位和刺激后时间直方图。 本文以电生理学方法揭示,猫小脑后叶第Ⅶ小叶皮层-核团投射存在较明确的纵区分布模式,纵区之间的分界线走向有一定的弯曲,与前叶略有不同。小脑后叶皮层从中线到两侧2.8mm为顶核区(FZ);其外侧为间位核区(IZ),最大宽度约为3.5mm;齿状核区(DZ)约始于5.0mm处。这三个不同纵区的PC对外周自然屈腕刺激都有反应,但反应细胞的百分数不同,FZ有59％的PC对外周刺激有反应,IZ为84％,DZ为20％。这些结果表明后叶第Ⅶ小叶具有类似于前叶的功能分布,IZ的PC对外周刺激有更大的调制作用,提示该皮层-核团投射的纵区结构有其特定的功能意义。     

长根小奥德蘑双孢菌株与四孢菌株核相变化的比较

用双苯并咪唑（Hoechst 33258）染色法分别对长根小奥德蘑Oudemansiella radicata双孢菌株和四孢菌株的菌丝、子实体、担孢子进行染色观察,结果表明：双孢长根小奥德蘑菌丝细胞多为单核,无锁状联合;原担子中单核进行一次有丝分裂形成两个横向或纵向排列的子核,这2个子核分别进入2个担孢子中,留下无核的空担子;成熟担孢子具有一个核。四孢长根小奥德蘑菌丝细胞大多数为双核,具有锁状联合;进入原担子中的两个单倍性细胞核先发生核配,形成一个二倍性的核,再经过减数分裂形成四个染色体减半的单倍性子核,     

我国西藏小反刍兽疫病毒China／Tib／Gej／07—30核衣壳蛋白基因和基因组启动子区的分子特征分析

首次对我国西藏小反刍兽疫病毒China/Tib/Gej/07-30的核衣壳蛋白(N)基因和基因组启动子(GP)区进行序列测定和分子生物学特征分析。首先应用逆转录聚合酶链式反应从发病山羊病料中扩增出小反刍兽疫病毒N基因片段,用cDNA3′末端快速扩增方法获得基因组启动子区片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析,绘制系统发生树。我国西藏小反刍兽疫病毒China/Tib/Gej/07-30的N基因由1689个核苷酸组成,编码525个氨基酸,与India/Jhansi/03等6个已知N基因全序列的PPRV毒株核苷酸和氨基酸序列同源性分别为91.7～97.6和94.9～98.5。小反刍兽疫病毒China/Tib/Gej/07-30N蛋白与磷蛋白作用的结构序列之一为495LFRLQAM501保守序列,N蛋白281-289位氨基酸含有一个T细胞表位,为281YPALGLHEF289保守序列。小反刍兽疫病毒China/Tib/Gej/07-30的GP区由107个核苷酸组成,与Tur-key2000等5株其他PPRV毒株同源性为91.8～98.2。N基因核苷酸序列和相应的氨基酸序列系统进化分析表明小反刍兽疫病毒China/Tib/Gej/07-30与亚洲国家分离株关系最近。     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. II. Phospholipids of ciliary and other membranes

The phospholipids of cilia and deciliated bodies of Paramecium tetraurelia were isolated and characterized. 1-alkyl-2-acyl-sn-glycero-3-(2′-aminoethyl) phosphonate (GAEPL), phosphatidylethanolamine, and 1-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (GPC) were the major lipids of Paramecium, and the minor lipids included phosphatidylinositol, cardiolipin, ceramide-(2-aminoethyl) phosphonate (CAEP), ceramide phosphorylethanolamine (COPE) and four sphingolipids whose identity was not established. The deciliated bodies contained 4% cardiolipin, 15% GAEPL, 41% phosphatidylethanolamine, 30% GPC and 3% each of CAEP and phosphatidylinositol; the cilia contained no cardiolipin, 24% GAEPL, 37% phosphatidylethanolamine, 15% GPC, 15% CAEP, 3% phosphatidylinositol, 2% COPE and small amounts (approx. 1%) of the four uncharacterized sphingolipids. No alteration in phospholipid composition was found among cells harvested in the various stages of growth. The phospholipids of six Paramecium mutants of three distinct phenotypes (pawn, paranoiac and fast) were also examined. Only one significant difference was found on comparison of the whole cell, deciliated body and cilia fraction of the mutants with the analogous fractions from wild type cells: the fast mutant, fA 97, had two extra, minor phospholipids (approx. 2%) in the deciliated body fraction that were tentatively identified as 1,2-diacyl-sn-glycero-3-(2′-aminoethyl) phosphonate (AEPL) and 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine (GPE).     

A kinome of 2600 in the ciliate Paramecium tetraurelia

Protein kinases play a crucial role in the regulation of cellular processes. Most eukaryotes reserve about 2.5% of their genes for protein kinases. We analysed the genome of the single-celled ciliate Paramecium tetraurelia and identified 2606 kinases, about 6.6% of its genes, representing the largest kinome to date. A gene tree combined with human kinases revealed a massive expansion of the calcium calmodulin regulated subfamily, underlining the importance of calcium in the physiology of P. tetraurelia. The kinases are embedded in only 40 domain architectures, contrasting 134 in human. This might indicate different mechanisms to achieve target specificity.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca²⁺ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca²⁺-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca²⁺, Sr²⁺, or Ba²⁺, but not by Mg²⁺ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24°C are transferred rapidly to 32°C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)⁺ RNA is 24°C and 35% at 32°C. Kinetic analysis of cDNA-poly(A)⁺ RNA hybridizations reveals three abundance classes of poly(A)⁺ RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24°C and 13000 at 32°C. The intermediately abundant and highly abundant classes are represented by 100–200 and 1–3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)⁺ RNA with increase in temperature.     

Fluctuation of the SP/non-SP phenotype in the C6 glioma cell line

Using the C6 glioma cell as a paradigm, we found that (i) the clonogenicity of C6 cells is several orders of magnitude higher than the percentage of SP cells; (ii) non-SP cells are able to generate SP cells, and conversely SP cells generate non-SP cells; (iii) non-SP sorted cells behave as tumorigenic cells. Hence, in C6 cells cultured in serum-containing medium, SP cells can be generated from non-SP cells. This dynamic equilibrium explains in C6 cells the maintenance of the SP phenotype with cell passaging and demonstrates the existence of tumorigenic non-SP cells.