四膜虫S1有性生殖的电镜观察 Ⅰ.小核的成熟分裂和核内微管系统

关于本株四膜虫的接合生殖,包括接合前的行为、减数分裂、核的分化以及接合后体的发育等,我们曾进行过光学显微镜的观察(陈阅增等1982)。我们发现本株四膜虫在接合前有聚集成团的现象,克隆内接合形成的接合后体能正常分裂,并产生正常的大核系     

一个自配型梨形四膜虫的接合及其细胞学过程

自从Elliott和Nanney(1952)描述了四膜虫(Tetrahymena sp.)的接合过程以后,国外对于四膜虫的接合型(mating types)特性以及接合的细胞学过程做了很多研究。Nanney(1953),Elliott和Hayes(1953)分别研究了自配型(selfers)和接合型梨形四膜虫(T.pyriformis)的接合过程。Elliott和Nanney都曾指出,自配型四膜虫的两接合体(Conjuga-nts)在完成核交换后不能分开,即使人力强使分开,二接合体也将解体而不能存活。Elliott(1973)认为,自配型四膜虫接合产生的后代不能存活这一特性,是四膜虫种群排除不稳定的自配型的一种途径。     

THE FATE OF MITOCHONDRIA DURING AGING IN TETRAHYMENA PYRIFORMIS

During the growth cycle of Tetrahymena pyriformis the mitochondria undergo changes in position, number, and structure. Ciliates in the logarithmic growth phase possess elongated mitochondria which are aligned along the plasma membrane and are closely associated with the kinetosomes and kinetodesmata. Mitochondria appear to divide across the long axis at this time, resulting in two or more products. Throughout this phase of growth mitochondrial divisions keep pace with cytokinesis so that the population of mitochondria remains at essentially the minimal level. As the ciliates enter the stationary growth phase the mitochondria increase in number, become oval to spherical in shape, and some migrate into the cytoplasm. Intramitochondrial masses of various configurations appear at this time. Some of the mitochondria lying in the cytoplasm become incorporated into vacuoles. Within these vacuoles either a single mitochondrion appears or several mitochondria may be seen along with other cytoplasmic structures. Later in the stationary growth phase the contained mitochondria are dense and the tubules are more compact than normal. Various stages in disorganization of the mitochondria are observed in a single large vacuole. Cytochemical tests reveal the presence of acid phosphatase, suggesting that hydrolysis of the vacuolar contents occurs. Lipid droplets increase in number during the middle and late stationary phase of growth. These events are interpreted as being associated with the normal process of aging in T. pyriformis.     

IDENTIFICATION OF MARINE DINOFLAGELLATES USING FLUORESCENT LECTINS1

Toxic and nontoxic species of marine dinoflagellates were characterized using fluorescent lectins. Lectin binding was detected by epifluorescence as well as spectrofluorometry. The binding assay of fluorescent lectins readily differentiated between morphologically similar species (i.e the toxic dinoflagellate Gymnodinium catenatum and the nontoxic Gymnodinium sp.). Lectins appear to be a useful tool to distinguish among different clones of the same species and, thus, possibly as a tool in dinoflagellate identification. Moreover, the lectins used show that thecate species have more binding sites and diversity in glycan moieties than athecate species.     

WST-8比色法检测梨形四膜虫细胞活性

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四膜虫S1有性生殖的电镜观察Ⅱ.接合区的形态与配子核的交换

四膜虫接合膜上的小孔是两接合体细胞质相连的通道。配子核形成后,按合膜由于增生而出现装有细胞器等的囊状折叠,并可脱离下来而落入另一细胞中,这可能是胞质交流的又一途径。配子核的交换不是从膜上原有小孔通过的,而是在溶酶体等作用下、使膜破裂,由核后方的微管推动进入对侧细胞中。 本文记述了四膜虫S1有性生殖过程中接合区的形态和配子核的交换。     

上海四膜虫大核类似程序性死亡的诱导

足叶乙甙可以诱导上海四膜虫大核在形态和生化方面出现类似程序性死亡的典型特征,如染色质凝集成颗粒状;分散的核仁连接成串;ＤＮＡ凝胶电泳出现阶梯状条带;彗星电泳出现慧尾。这就提示,利用足叶乙甙诱导四膜虫大核发生类似程序性死亡是一个有用的实验模式,它可能对阐明在自然状态下大核类似程序性死亡的机理有启发和借鉴意义。同时也提出了染色体形态特征变化和彗星电泳有可能作为鉴定四膜虫大核发生类似程序性死亡的指标。     

THE SYNTHESIS OF MICROTUBULE AND OTHER PROTEINS OF THE ORAL APPARATUS IN TETRAHYMENA PYRIFORMIS

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.     

EFFECTS OF DIPEPTIDES CONTAINING THE AMINO ACID,PROLINE ON THE CHEMOTAXIS OF TETRAHYMENA PYRIFORMIS. EVOLUTIONARY CONCLUSIONS ON THE FORMATION OF HORMONE RECEPTORS AND HORMONES

Our investigations demonstrate that proline-containing dipeptides can provoke a chemosensory response from the unicellular Tetrahymena pyriformis The chemotactic effects of the dipeptides have a close relationship with the side chain and the lipophilicity of the amino-terminal amino acid. Comparison of ‘mirror’ variants of proline-containing dipeptides points to the fact that dipeptides with small side chain and non-polar character amino acids (Gly-Pro, Ala-Pro) are preferred on the amino-terminal end. In the case of amino acids with very variable side chains, small (Pro-Gly) and the large side chain and non-polar character amino acids (Pro-Leu, Pro-Phe) on the carboxyl-terminal end can induce significant chemotactic responses. With valine on any terminus the proline-containing dipeptide induced a weak repellent effect.     

THE MECHANISM OF THE WATER EXPULSION VESICLE OF THE CILIATE TETRAHYMENA PYRIFORMIS

Analysis of high-speed (150 frames/sec) cinematographs of the filling and expulsion of the water expulsion vesicle of Tetrahymena pyriformis shows that the vesicle fills as water is pumped into it by contractions of at least four ampullary sacs which are continuous with the endoplasmic reticulum. When filled, the vesicle is pressed against its two excretory pores by cyclotic movements of the cytoplasm. This pressure closes the apertures of the ampullae, preventing backflow from the vesicle into them, and also spreads the pellicle of and at the pore, thereby stretching and rupturing the pore-sealing membrane. The vesicle is then invaginated by the cytoplasmic pressure, driving fluid out of the pore. The pore-sealing membrane then reforms, apparently by constriction, and the vesicle is again filled. Electron micrographs show that crisscrossed pore-microtubules extend from the pore to the openings of the ampullae, anchoring the vesicle in place. Each pore is surrounded by a stack of at least 11 ring-microtubules, to which the anchoring pore-microtubules are attached. The pore-microtubules appear to exert tension which assists in spreading the pore, aiding cyclotic pressures in rupturing the pore-sealing membrane. A possible mechanism for the cyclotic pressure and ampullary contraction is proposed.     

THE EFFECTS OF HIGH HYDROSTATIC PRESSURE ON THE MICROTUBULES OF TETRAHYMENA PYRIFORMIS

Exposure of Tetrahymena pyriformis to 7,500 or 10,000 psi of hydrostatic pressure for 2, 5, or 10 min intervals results in a change in cell shape and ciliary activity. Shape changes occur concurrently with a degradation of longitudinal microtubules in a posterior to anterior direction. High pressure also causes a disruption of ciliary activity. Fine structural analysis reveals a breakdown (presumably microtubule depolymerization) of the central ciliary microtubules. The depolymerization begins at the junction of the central ciliary microtubules with the axosome and progresses distally along the ciliary shaft for a distance of about 0.5 µ.     

上海四膜虫的胞质配合

对上海四膜虫（Ｔｅｔｒａｈｙｍｅｎａ ｓｈａｎｇｈａｉｅｎｓｉｓ）有小核细胞和无小核细胞的接合过程进行了跟踪观察,发现在四膜虫中也存在胞质配合（ｃｙｔｏｇａｍｙ）。在接合对中,有小核细胞一方可以完成正常的减数分裂过程,既有配子核的形成、配子核的融合、配子核分裂,最终形成两个有小核的子细胞。虽然有小核细胞的配子核并不进入无小核细胞,但是两细胞间却有细胞的迁移与交换。在整个接合过程中,无小核细胞的大核     

THE NUCLEIC ACIDS OF BASAL BODIES ISOLATED FROM TETRAHYMENA PYRIFORMIS

Pellicular fragments were isolated from ethanol-fixed cells of the holotrichous ciliate Tetrahymena pyriformis by the action of digitonin. The isolated pellicles were further fragmented and the basal bodies of the cilia isolated from them by three methods. The preparations, examined in the electron microscope as embedded sections or negatively stained samples, consisted mainly of somewhat deformed pellicular material, the bulk of which was basal body. DNA was determined by the diphenylamine method and by reaction with DNase, and RNA, by the orcinol method. Nucleic acids were isolated by phenol extraction and analyzed spectrophotometrically and by reaction with RNase. The assays indicated 1.2 to 2.6 per cent RNA, similar to previously published work, but only 0.0 to 1.0 per cent DNA, near enough the sensitivity limits to render the presence of DNA in the preparations uncertain. Although the isolation procedure removed nuclear contents and ribosomes, the nucleic acids could still be a residual contaminant bound to the pellicle during the isolation. Hypotheses of basal body self-duplication, moreover, can be constructed both with and without nucleic acids.     

A TEMPERATURE-COMPENSATED ULTRADIAN CLOCK OF TETRAHYMENA: OSCILLATIONS IN RESPIRATORY ACTIVITY AND CELL DIVISION

Both a circadian clock and an ultradian clock (period 4—5 h) have previously been described for the ciliated protozoon Tetrahymena. The present communication demonstrates the existence of yet another cellular clock: an ultradian rhythm with a period of about 30 min. The period was found to be well temperature-compensated over the range studied, i.e., between 19°C and 33°C. Ultradian rhythmicity was initiated by dilution of stationary-phase cultures, which were kept previously in a light-dark cycle, into fresh medium. LD treatment during stationary phase was an absolute requirement, since cultures kept in either LL or DD did not produce the ultradian rhythmicity after refeeding. The clock exerts control over respiration; the observed oscillation in oxygen uptake is just a hand of the clock: after a limitation of oxygen supply had ended, the rhythm resumed with the same phase and period as that in control cultures. The clock exerts temporal control also over cell division; in the refed culture cell division resumed with an oscillation in the number of dividing organisms. The period of this oscillation corresponded to that of the rhythm in respiratory activity, indicating that the same ultradian clock may exert control over different cellular functions. Analysis of a second Tetrahymena strain indicates that period length of the ultradian clock is a strain-specific characteristic.     

THE EFFECT OF 5-BROMODEOXYURIDINE ON DNA REPLICATION AND CELL DIVISION IN TETRAHYMENA PYRIFORMIS

Populations of Tetrahymena pyriformis were grown in a chemically defined medium containing the thymidine analogue 5-bromodeoxyuridine (BUdR). About 65% of the thymidine sites in DNA were substituted by BUdR. During the first generation in the presence of BUdR, all DNA became hybrid. After the following cell division, in about 80% of the cells the second DNA replication round was initiated but no further cell division took place. The cells could be rescued by removing BUdR and adding thymidine. New replication took place before the first cell division. However, although the cells contained double heavy as well as hybrid DNA, only the hybrid DNA was replicated. After a full replication of the hybrid DNA, normal growth was restored. Melting profiles of normal, hybrid, and double heavy DNA indicated a structural change of the double heavy DNA.     

THE FINE STRUCTURE OF THE NUCLEI OF TETRAHYMENA PYRIFORMIS THROUGHOUT THE CELL CYCLE

The fine structure of the nuclei of logarithmically growing Tetrahymena pyriformis, strain HSM, was studied at 30-minute intervals throughout the cell cycle. Organisms were selected at similar stages of cytokinesis by means of a braking pipette, incubated, fixed in OsO₄, and embedded in agar to facilitate subsequent preparation for electron microscopy. Aggregates of micronuclear chromatin underwent a decrease in density and number with a concomitant increase in size throughout interphase. There were no impressive changes in macronuclear morphology. It was found possible to estimate a cell's progress through interphase by observation of micronuclear morphology, but attempts to correlate changes in fine structure with periods of DNA synthesis were unsuccessful.     

SIMULTANEOUS SYNTHESIS OF HISTONE AND DNA IN SYNCHRONOUSLY DIVIDING TETRAHYMENA PYRIFORMIS

Histone and DNA syntheses have been studied in synchronously dividing Tetrahymena pyriformis GL. During the heat treatment necessary to synchronize cultures of this amicronucleate protozoan, the DNA content of the already polyploid macronucleus increases. When the cells begin synchronous division, their DNA content is reduced in a stepwise process which is closely paralleled by reduction of macronuclear histone content. During cell division, the contents of DNA and histone decrease by slightly more than twofold, and in the subsequent S phase, DNA and histone increase simultaneously to 85% of the values expected if all chromosomes were to double. The first step in the process of reduction of DNA and histone contents is their decrease in excess of twofold, and this is accomplished by removal of extrusion bodies from the nuclei of dividing cells. The second step is a mechanism which allows, in effect, only 70% of the chromatin in the average nucleus to duplicate. Such partial duplication suggests that both histone and DNA syntheses in synchronous Tetrahymena depend upon a regulatory mechanism, the mediating elements of which are localized in only certain chromosomes.     

TIME- AND CONCENTRATION-DEPENDENCE OF THE GROWTH-PROMOTING ACTIVITY OF INSULIN AND HISTAMINE IN TETRAHYMENA. APPLICATION OF THE MTT-METHOD FOR THE DETERMINATION OF CELL PROLIFERATION IN A PROTOZOAN MODEL

The unicellular ciliateTetrahymena pyriformiswas treated with different concentrations of insulin or histamine and at different time points the cell density was measured, using a tetrazolium-based semiautomated colorimetric assay (MTT). The assay was suitable to determine the rate of cell proliferation of Tetrahymena. Insulin in each concentration significantly elevated the cell count up to 3h. After that, it was neutral or its effect was insignificant. Histamine at 10^?5m concentration diminished cell count at 3, 5, 7 and 24h. At 10^?6m concentration there was no difference and at 10^?7m concentration it enhanced cell division up to 5h, after that there being no difference. The two hormones have cell division promoting activity for cells of higher animals and the experiments demonstrate this effect already at a unicellular level.