原生动物贻贝棘尾虫微管胞器的荧光标记与显示

采用FLUTAX直接荧光标记和抗α微管蛋白抗体的间接免疫荧光标记显示,原生动物贻贝棘尾虫(Stylonychia mytilus)细胞微管胞器由口围带、波动膜、额腹横棘毛、左右缘棘毛、背纤毛等纤毛器微管骨架、纤毛器基部附属微管和其他皮层微管骨架组成。纤毛器微管骨架和基部附属微管按皮层纤毛模式定位;皮层左、右侧微管带和领肋壁微管等其他皮层微管构成细胞特定位置的皮层微管骨架,并可能为具有背腹分化的腹毛目纤毛虫所特有,对维持细胞背腹面的形态、支持附近纤毛器(如左、右缘棘毛)的运动起作用。本文较完整地阐述了其细胞骨架的三维构形,对于深入了解纤毛虫细胞微管骨架的结构和分布特征,进一步揭示微管类胞器的功能是有意义的。     

Fine Structure of the Macronuclear Anlage of the Hypotrichous Ciliate Keronopsis rubra

In exconjugants of the hypotrich Keronopsis a large, highly polyploid macronuclear anlage is formed from which condensed chromatin bodies are passed into the cytoplasm where they are thought to give rise to the numerous small macronuclei of the vegetative cell. Electron microscopy shows that the chromatin bodies within the macronuclear anlage are separated from each other by sheets of low contrast lamellar material. The anlage appears therefore as a composite nucleus containing prepacked units which are extruded into the cytoplasm following condensation.     

棘尾虫酸性磷酸酶的定位及诱导表达

本文利用电镜对贻贝棘尾虫(Stylonychia mytilus)体内酸性磷酸酶的定位进行了观察。发现其体内除含有溶酶体性的酸性磷酸酶(ACPase)外，在纤毛内部还存在非溶酶体性的ACPase。另外，本文还利用光镜和酶细胞化学技术观察并比较了棘尾虫年轻态和衰老态个体体内ACPase在食物诱导下的表达量随时间发生的动态变化和差异，印证了棘尾虫个体的衰老也是与细胞内的酶的活性和含量的降低相伴随的[动物学报49(2)：218—223，2003]。     

食物在贻贝棘尾虫(原生动物,纤毛虫)细胞内的消化过程

利用免疫荧光显微术、透射电镜及电镜酶细胞化学技术对食物在腹毛类纤毛虫-- 贻贝棘尾虫细胞内的消化过程进行了追踪观察.食物在进入消化腔隙的初期,外面包被着一层膜结构,但此外层膜很快被消化而消失;尔后食物在贻贝棘尾虫体内的消化过程可分为两种方式:一种方式为直接在消化腔隙内完成,此过程同1982年Kaul et al.和Das s et al.的报道;而另一部分食物的消化过程表现为食物逐步向细胞质内突入,以致最终完全被细胞质包围形成一被膜包裹的圆泡状结构;在此过程中,细胞质边缘突出众多含有酸性磷酸酶等物质的小囊泡结构,小囊泡与食物融合,其内的酸性磷酸酶等水解酶释放出来, 食物的表膜逐渐瓦解,个体逐渐变小,最终消化后的营养物质被释放到细胞的消化腔隙内; 这种食物通过胞口与胞咽达消化腔隙后,再由细胞质将其包裹形成的食物泡,我们称之为" 后成食物泡".这两种消化方式与贻贝棘尾虫的消化胞器为一腔隙结构相适应[动物学报 5 4(3):510-516,2008].     

A PCR-based method to distinguish the sibling species Stylonychia mytilus and Stylonychia lemnae (Ciliophora, Spirotrichea) using isocitrate dehydrogenase gene sequences

A differentiation, based on morphological characters, between Stylonychia mytilus and Stylonychia lemnae is very difficult, especially for non-specialists. These two sibling species were considered as one species, S. mytilus, until detailed cytological and genetic studies could show the existence of two genetically isolated varieties. Further morphological and biochemical analyses verified the separation and finally in 1983 a new species S. lemnae was described. The examination of several isoenzymes revealed unambiguous differences in the banding pattern of isocitrate dehydrogenase (IDH) between these two species. Therefore, the IDH gene of 30 isolates of S. lemnae and S. mytilus coming from various regions all over the world were amplified and sequenced. The sequence analyses revealed intraspecific as well as interspecific substitutions, which were used for the development of species-specific PCR primers for both species. Application of these species-specific primer pairs now allows a very easy and clear identification of both sibling species.     

Characterization of the Life Cycle and Heteromeric Nature of the Macronucleus of the Ciliate Chilodonella uncinata Using Fluorescence Microscopy

Only a limited number of studies exist on the life cycles of nonmodel ciliates such as Chilodonella uncinata (Cl: Phyllopharyngea). The handful of papers on this taxon indicate the presence of a heteromeric macronucleus, marked by separate DNA‐rich and DNA‐poor regions. Here, we study the life cycle of C. uncinata using confocal laser scanning microscopy with 4′,6‐diamidino‐2‐phenylindole staining, which allows us to differentiate nuclear dynamics of the micronucleus and the macronucleus during life‐cycle stages. We photo‐documented various stages and confirmed aspects of the development of the new macronucleus previously characterized by electron microscopy. We further reveal the heteromeric structure of the macronucleus with Z‐stacks and three‐dimensional (3D) reconstructions. We find no evidence for the presence of an endosome at the center of the macronucleus during vegetative growth. In addition to illustrating the life cycle of this ciliate, the approaches developed for this study will enable additional comparative analyses of nuclear dynamics using fluorescence microscopy.     

Fluorescence in situ hybridization with specific oligonucleotide rRNA probes distinguishes the sibling species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea)

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species.     

The Structure and Development of the Dorsal Bristle Complex of Oxytricha fallax and Stylonychia pustulata*

SYNOPSIS. Oxytricha fallax and Stylonychia pustulata possess 6 rows of dorsal bristle units. Each dorsal bristle unit consists of a pair of kinetosomes; the anterior kinetosome has a cilium and the posterior kinetosome a ciliary stub. The kinetosome pair, located at the bottom of a cortical pit surrounding the cilium and ciliary stub, is surrounded by an asymmetrical fibrillar mass. Future rows 1-4 are formed from 2 sets of primordia originating within mature dorsal rows 1-3. Rows 5 and 6 originate from the anterior regions of both right marginal cirral primordia. Old dorsal bristle units utilized in formation of primordia are presumably maintained in the new rows of the proter and opisthe; those outside the primordia are resorbed. The morphogenetic pattern of the Oxytrichidae is similar to those of the Urostylidae and Holostichidae, but quite different from that of the Euplotidae.     

用DNA银染法研究传染性软疣病毒的形态发生发育过程

用ＤＮＡ银染技术显示了传染性软疣病毒（ＭＣＶ）形态发生发育及其过程中ＤＮＡ的变化。结果显示：在被感染的皮肤表皮细胞内都有一个大小及构型不同的银染区（病毒工厂）。ＭＣＶ的发生发育均在银染区内而不在胞质区内。其发生过程是先在细胞一侧的胞质内复制合成病毒ＤＮＡ等物质,形成中等密度的银粒大小不等的银染区（病毒前包涵体区）,然后在其中形成致密的细粒状银染区（病毒前基质区）,接着在后者的周围出现弧形的粗粒银沉淀（初期ＭＣＶ的外膜）,逐渐分割包围病毒前基质而形成初期ＭＣＶ。在发育过程中,初期ＭＣＶ的外膜、基质,核心外膜及核心均经过一系列的形态变化。侧体是中期ＭＣＶ向成熟期发育中出现的暂时性结构,其本质为含ＤＮＡ成分的病毒基质。本研究提示：ＭＣＶ的ＤＮＡ物质进入皮肤表皮细胞后能大量复制,合成大量的病毒蛋白质,自主地装配成完整的初期ＭＣＶ,后者有独特的形态发育过程。     

An analysis of the development program in relation to RNA metabolism in the ciliate Stylonychia mytilus

In the ciliate Stylonychia during conjugation and the terminal stages of the division cycle, development is dependent upon presynthesized mRNA. The conjugating animals show complex nuclear and cortical changes in a chronological order. Disturbance of the cortical organization or arrest of nuclear divisions in conjugants and exconjugants does not induce the cells to adjust the developmental program; the nuclear and cortical changes proceed without any reference to each other in a defined course. Similarly the terminal stages of the division cycle are not affected by cortical amputations. In contrast, vegetative animals up to mid S phase show constant RNA turnover in relation to the progress of the division cycle. In these animals, cortical amputation leads to an immediate arrest of the cell-cycle events, which are resumed only after regeneration. Nucleocytoplasmic interactions in relation to RNA metabolism of the ciliate are discussed.     

Morphological study of bacteria of the respiratory system using fluorescence microscopy of Papanicolaou-stained smears with special regard to the identification of Mycobacteria sp.

In Papanicolaou-stained smears certain structures such as nucleoli, Pneumocystis carinii , Charcot-Leyden crystals, bacteria and fungi show a brilliant fluorescence. the morphological characteristics of microorganisms which can be detected by this system, especially mycobacteria, are described. This screening method offers the possibility of providing the clinician with a provisional diagnosis within hours. Proof of the nature of the organisms should be obtained by culture.     

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000–7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus–homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed. Dev. Genet. 21:201–211, 1997.© 1997 Wiley-Liss, Inc.     

Effects of Glutaraldehyde and Osmium Tetroxide on Hypotrichous Ciliates, and Determination of the Most Satisfactory Fixation Methods for Electron Microscopy

SYNOPSIS. In electron microscope studies on hyppotrichous ciliates, cytolysis and/or body deformation–resulting from insufficient contact with glutaraldehyde and poor infiltration of Epon 812, particularly into the buccal cavity, usually were observed. Fixation experiments were carried out to examine the effects of some fixatives on Euplotes eurystomus, Oxytricha bifaria and Stylonychia mytilus to establish the best fixation technic applicable to all species of hypotrichous ciliates. Although the effects of fixation varied considerably with the species, 2 fully satisfactory fixation methods were developed by using OsO₄ and glutaraldehyde. In one, a mixture of both fixatives was employed; in the other a very short application of OsO₄ was followed by glutaraldehyde. The problem of infiltration was solved by using Spurr's low-viscosity embedding medium in place of Epon 812.     

Microwave Irradiation for Rapid and Enhanced lmmunohistochemical Staining: Application to Skin Antigens

We describe a method in which microwave irradiation is used to reduce substantially the incubation time for immunoperoxidase staining of antigens in cryostat sections of pso-riatic skin. An incubation time of 5-9 min irradiation at 80 W generated similar or better staining intensity for all antibodies used compared to the standard methods using 30-60 min incubation at room temperature. Although we found that microwave irradiation could be used with all antibodies tested, independent of whether they recognized extracellular, membrane or cytoplasmic antigens in skin, the conditions needed to be optimized for each antibody.     

Fixation and Staining of Planaria for Histological Study

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acld-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.     

A Mixture of Paraphenylenediamine and Imidazole: Its Effect on the Extraction of Lipid Droplets during Electron Microscopy Staining

To prevent extraction of lipids during a double staining procedure for electron microscopy, the tissue slices, double fixed with glutaraldehyde and osmium tetroxide to preserve microvesicular lipid droplets in the cytoplasm, were immersed for 2 hr in veronal buffer (pH 9.0) containing 0.5% p-phenylenediamine and 0.5% imidazole immediately after postfixation. The stained sections of the immersed tissue slice showed blackened, well circumscribed lipid droplets similar to those in corresponding unstained sections. Moreover, highly contrasting features of the cellular architecture could be visualized with the double stained, as well as routinely prepared sections.     

Immunohistochemical Double Staining with Immunogold-Silver and Alkaline Phosphatase to Identify Nuclear Markers of Cellular Proliferation

To facilitate cell kinetics studies of brain tumors labeled with thymidine analogs, we developed a new method to identify nuclei labeled sequentially with bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) by double staining with immunogold-silver and alkaline phosphatase. Patients received an intraoperative infusion of BUdR: excised tumor specimens were immediately labeled with IUdR in vitro. fixed with 70% alcohol, embedded in paraffin, and cut into 6 pm sections. The sections were incubated first with BR-3. a monoclonal antibody that recognizes only BUdR, and then with IU-4. a monoclonal antibody that recognizes both BUdR and IUdR: sections were counterstained with hematoxylin to identify unlabeled nuclei. Nuclei labeled only with IUdR stained red, whereas those labeled with BUdR or with both BUdR and IUdR stained black against a red background: unlabeled nuclei stained blue. This method was the most efficient differential staining technique to identify nuclei labeled only with IUdR and those labeled with BUdR among unlabeled nuclei.     

糖蛋白PAGE分离后的糖基显色法

鸡卵清蛋白通过SDS-PAGE分离后,用过碘酸-希夫(PAS)显色法可以使卵清蛋白显红色,而用糖苷酶去除卵清蛋白的糖基可以去除相应的红色。考马斯亮兰染色结果显示,去糖基后的卵清蛋白分子量略有减小。可见,PAS可方便地使糖蛋白呈现红色。     

Commitment to the First Cortical Reorganization During Conjugation in Stylonychia mytilus: An Argument for Homology with Cortical Development During Binary Fission

The asexual nature of the first cortical reorganization of conjugation in Stylonychia was analyzed by comparing the effect of amputation performed at different stages of early conjugation to that performed on vegetative cells at different stages of the cell cycle. Amputation of vegetative cells delineated a point of commitment to binary fission at 0.51–0.57 of the cell cycle. Cells amputated before this point were induced to undergo the regenerative mode of asexual development, but those amputated after this point continued with binary fission. In parallel, during conjugation a similar commitment was made around the time of formation of tight mating-pairs: early conjugants amputated around this time might undergo regeneration, and those operated on after this stage continued with the first cortical reorganization as in typical conjugants. The two mates of a pair might differ in their response to amputation, suggesting that the timing of commitment to the first cortical reorganization is not related to the events of conjugation, but rather is individually determined in the vegetative cycle of the cells before they pair up in mating. These observations provide support for the notion that the first cortical reorganization of conjugants is homologous to the asexual mode of cortical development in dividers, according to the theory of developmental heterochrony in the sexual reproduction of hypotrichs. The timing of commitment to the first cortical reorganization was found to temporally correlate with the entrance of the micronuclei into meiosis. Since the first cortical reorganization can proceed without the micronucleus, this raises the possibility that initiation of micronuclear meiosis is closely coupled with, and may be determined by, the commitment to the first cortical reorganization.