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.     

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.     

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.     

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。     

The cytologic diagnosis of Mycobacterium kansasi tuberculosis by fluorescence microscopy of Papanicolaou-stained specimens

The cytologic diagnosis of Mycobacterium kansasi tuberculosis by fluorescence microscopy of Papanicolaou-stained specimens
The sensitivities of (i) Papanicolaou fluorescence, (ii) auramine rhodamine fluorescence, and (iii) Ziehl-Neelsen staining were compared for their ability to detect the atypical mycobacterium Myco. kansasi in cytological samples. Ninety-two cases were investigated, and the sensitivities of the three methods of detection were found to be 36.9%, 12.0%, and 20.7%, respectively. The control groups consisted of 30 specimens from cases of bronchial carcinoma and 30 of pneumonia. All cases were proved by microbiology. No false-positive results were recorded using Papanicolaou fluorescence. An important but coincidental finding arising from this study was that infection by the atypical mycobacterium Myco. kansasi causes cytological patterns corresponding to those normally associated with acute pneumonia and not to tuberculosis.     

Staining Paraffin Sections Without Prior Removal of the Wax

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10-20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

Anomalous Cases of Conjugation and Spontaneous Autogamy in Stylonychia mytilus: A Note on the Asexuality of Early Conjugants

Four types of anomalous conjugation were documented in Stylonychia mytilus. Type I pairs were formed between mates of different sizes. These pairs exhibited an abnormal site of fusion in at least one of the mates, and the mates might face each other ventrally throughout conjugation instead of the normal side-by-side position. Type I pairs underwent sexual nuclear development and proceeded with the first cortical reorganization as in normal conjugants. Type II involved pairing at the anterior ends of mates with ventral surfaces facing the same direction. These pairs also underwent sexual nuclear development. Hence, aberrant orientation of the mates, and also ectopic sites of cytoplasmic fusion, if extensive, would permit sexual development. Type III pairs were united ventral-to-ventral with their anterior-left sides at the adoral zone of membranelles, and remained as such throughout conjugation. In these pairs, nuclear and cortical events were typical of the asexual development of physiological reorganization. In Type IV pairs, one mate of the pair possessed a fission furrow and developed two sets of ciliature typical of binary fission, while the other mate might undergo physiological reorganization or binary fission. Type III and Type IV pairs thus reveal the asexual state of early conjugants, which can pursue either one of the two modes of asexual cortical reorganization; these cases reinforce the notion of overlap of asexual and sexual cycles during conjugation of hypotrichs. Spontaneous autogamy was documented for the first time for this genus. The autogamonts proceeded with nuclear development and with the first cortical reorganization. Some probably underwent second and third reorganizations, as in conjugants, but accompanied by abnormalities, particularly in the stages beyond fertilization. Post-autogamous clones were nonviable except for one dubious case.     

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.     

Bassam和Sanguinetti银染方法在SRAP和TRAP标记中的比较研究

银染作为一种重要的DNA染色方法,对分子标记检测有重要影响.SRAP标记和TRAP标记是两种较为新型的分子标记,近年来得到了广泛应用,尤其在缺少遗传图谱的物种中应用价值更大.以普通烟草种(Nicotiana tabacum L.)烤烟和香料烟品种作为供试材料,对Bassam和Sanguinetti两种银染方法,在标记检测效率、成本及染色效果等方面进行了研究,并对其在SRAP标记和TRAP标记中的应用进行了探讨.结果表明,Sanguinetti银染法比Bassam银染更为简便、经济,染色照片的色阶图说明Sanguinetti染色方法的背景与扩增带区分明显,能够清楚地读带;且该方法能够扩增出很好的SRAP和TRAP标记谱带.因此,推荐在SRAP和TRAP标记检测中采用Sanguinetti银染方法.     

Detection of Charcot-Leyden crystals by fluorescence microscopy of Papanicolaou-stained smears of sputum, bronchoalveolar lavage fluid, and bronchial secretions

Fluorescence microscopy was used to examine Papanicolaou-stained smears of sputum and other secretions from the respiratory tract. Under these conditions Charcot-Leyden crystals (CLC) appear as bright yellow-green fluorescing needles. the study was performed to determine the value of this approach for the diagnosis of allergic lung diseases. the time taken to detect the crystals was recorded and the sensitivity of fluorescence microscopy for the detection of CLC was compared with light microscopy of the same samples. the data show that fluorescence microscopy is superior to light microscopy for the detection of CLC. the characteristic needle-shaped crystal can be recognized easily and fragments of crystals could be easily identified. In doubtful cases of allergic lung diseases, fluorescence microscopy may be used to supplement light microscopy for the detection of Charcot-Leyden crystals.