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。     

Hoechst 33342-induced autophagy protected HeLa cells from caspase-independent cell death with the participation of ROS

Abstract

Autophagy, an evolutionarily-conserved intracellular organelle and protein degradation process, may exhibit drastically different effects on cell survival depending on the particular environmental and culturing conditions. Hoechst 33342 (HO), a fluorescent dye widely used for staining DNA, has been reported to induce apoptosis in mammalian cells. Here we showed that, in addition to caspase-independent cell death, HO also induced autophagy in HeLa cells, as evidenced by the accumulation of autophagosomes, LC3 form conversion and LC3 puncta formation in a cell line stably expressing GFP-LC3. HO treatment led to generation of reactive oxygen species (ROS), and inhibition of ROS with N-acetyl-l-cysteine (NAC) abrogated both autophagy and caspase-independent cell death. Finally, autophagy played a protective role against caspase-independent cell death, as cell death induced by HO was enhanced under pharmacological and siRNA-mediated genetic inhibition of autophagy.     

Motility Events of Trichocyst Insertion in Paramecium tetraurelia*

SYNOPSIS. Following electroshock-induced extrusion of its inserted trichocysts, Paramecium tetraurelia rapidly begins replacement of the population of lost organelles. Light microscopy of the cortical insertion of new trichocysts reveals a series of characteristic motility activities. An uninserted trichocyst in the cyclotic flow of the cell appears to be “captured” and removed to the noncyclotic, subcortical regions. The trichocyst then makes a series of saltatory motions which apparently serve to transport it to the cortex, with proper orientation (tip first) for insertion. Trichocyst saltations end with either cortical insertion of the organelle, or return to cyclosis. If the trichocyst is inserted, it makes a series of unique pivoting movements around the motionless tip. This form of motility, termed “wobble,” continues for a short period of time. After cessation of wobble, the insertion of the trichocyst is apparently complete, since no further motility is observed. With the aid of these observations it was possible to identify saltatory motility as the means for transporting trichocysts to the cortex for insertion, and also to observe a motility of unknown significance (wobble) apparently associated with the process of cortical insertion.     

Apoptosis in experimental myocardial infarction in situ and in the perfused heart in vitro

We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.     

Nuclear and Cortical Regulation in Doublets of Paramecium: II. When and How do Two Cortical Domains Reorganize to One?

Homopolar doublets with twofold rotational symmetry were generated in Paramecium tetraurelia and in P. undecaurelia by electrofusion or by arrested conjugation. These doublets underwent a complex cortical reorganization over time, which led to their reversion to singlets. This reorganization involved a reduction in number of ciliary rows, a progressive inactivation and loss of one oral meridian, and a reduction and eventual disappearance of one cortical surface (semicell) situated between the two oral meridians. The intermediate steps of this reorganization included some processes that resemble those previously described in regulating doublets of other ciliates, and others that are peculiar to members of the \"P. aurelia\" species-group and some of its close relatives. The former included a disappearance of one cortical landmark (a contractile vacuole meridian) and transient appearance of another (a third cytoproct) within the narrower semicell. The latter included a reorganization of the paratene zone and the associated invariant (non-duplicating) region to occupy the entire narrower semicell and a redistribution of zones of most active basal-body proliferation within the opposite, wider semicell. The final steps of reorganization involved anterior displacement, invagination, and resorption of one of the two oral apparatuses and eventual disappearance of the associated oral meridian. An oral meridian deprived of its oral apparatus, either by spontaneous resorption or microsurgical removal, could persist for some time in \"incomplete doublets\" before regulating to the singlet condition. The phylogenetically widespread events encountered in the regulation of doublets to singlets suggest that Paramecium shares some of the global regulatory properties that are likely to be ancestral in ciliates. The more specific events are probably associated with the complex cytoskeletal architecture of this organism and with the frequent occurrence of autogamy that was described in the preceding study (Prajer et al. 1999).     

Genetic Analysis of Trichocyst Discharge of the Wild Stocks of Paramecium tetraurelia*

SYNOPSIS. Aberrant discharge of trichocysts in response to picric acid occurs in 8 of the 28 wild stocks of Paramecium tetraurelia. There are at least 4 distinguishable phenotypes: nondischarge, stocks 139, 163, 169, and 242; temperature-sensitive nondischarge, stock 126; leaky nondischarge, stock 203; and a clonally unstable phenotype, stocks 146 and 148. From each of these stocks a single recessive gene causing nondischarge has been isolated by backcrosses to stock 51. The original stocks 126, 146, and 148 possess other genes which affect the extracted genes. The copper resistance locus is ～ 10 centiMorgans from nd169 and nd242, but none of the other nondischarge genes are linked to 6 marker loci. The genes nd169 and nd242 are only 0.5 centiMorgans apart making them the closest known pair of loci in P. tetraurelia. The genes nd126 and nd242 are distinguishable alleles at the same locus and the genes nd146 and nd148 are apparently identical alleles. The large number of loci involved in producing a similar phenotype in different stocks supports the idea that mutation is much more important than gene flow in this highly inbreeding species.     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

A Role of Calcium Ions in Chemical Induction of Mating in Paramecium tetraurelia*

SYNOPSIS. Conjugation in Paramecium tetraurelia can be induced within mating-reactive cultures of a single mating type by treating the cells with solutions of KC1 + acriflavine in culture medium low in Ca²⁺. Gene mutations with known physiologic effect were used as selective inhibitors of cell surface membrane function to see which functions are necessary for chemical induction of conjugation. The results strongly suggest that a transient increase in the internal concentration of calcium at the very beginning of chemical induction is a necessary but not sufficient step.     

Hoechst 33342 induces radiosensitization in malignant glioma cells via increase in mitochondrial reactive oxygen species

Abstract

Mitochondrial DNA plays an important role in cellular sensitivity to cancer therapeutic agents. Hoechst 33342, a DNA minor groove binding ligand, has shown radiosensitizing effects in different cancer cell lines. In the present study, the possible binding of Hoechst 33342 with mitochondrial DNA, isolated from human cerebral glioma (BMG-1) cells, was investigated and consequences of this binding on excessive reactive oxygen species (ROS) generation in irradiated BMG-1 cells were studied. Alteration in the fluorescence spectroscopic characteristics of Hoechst 33342 suggested binding of Hoechst 33342 with isolated mitochondria and mitochondrial DNA. Persistent increase in level of ROS in the presence of Hoechst 33342 has been observed, which was further enhanced in irradiated cells. Investigations using inhibitors of ETC complex I suggested that mitochondrial bound Hoechst 33342 contributed to increased ROS, which was associated with alteration in ΔΨm and antioxidant machinery. These factors appeared to contribute in potentiating radiation-induced cell death in BMG-1 cells. The finding from these studies will be useful in designing better anti-cancer strategies.     

Genetic analysis of mating type differentiation in Paramecium tetraurelia. III. A mutation restricted to mating type E and affecting the determination of mating type

In P. tetraurelia each cell is determined to express only one of the two complementary mating types, O and E. This determination is under cytoplasmic control and seems to be achieved only by the commitment or noncommitment to the expression of mating type E. All the previously known mutations affecting the differentiation of mating type prevent the expression of the E mating type (O-restricted mutations) without affecting the determination process. An E-restricted mutation was obtained: mtF^E. Its phenotypic properties indicate that the mutation affects the determination process itself. When an O cell becomes mtF^E/mtF^E it acquires the E mating type and an E-determining cytoplasm. We propose that this constitutive determination for the E mating type is due to the inefficiency of a factor which is normally active in an O cell. This factor would act like a repressor and stabilize the E functions under an inactive state.