THE ULTRASTRUCTURE OF MITOSIS AND CYTOKINESIS IN THE SARCINOID CHLOROKYBUS ATMOPHYTICUS (CHLOROPHYTA,CHAROPHYCEAE) REVEALED BY RAPID FREEZE FIXATION AND FREEZE SUBSTITUTION1

Mitosis and cytokinesis in vegetative cells of the sarcinoid green alga Chlorokybus atmophyticus Geitler were examined with rapid freeze fixation, freeze substitution, and transmission electron microscopy. The taxonomic placement of C. atmophyticus in the class Charophyceae sensu Stewart and Mattox is corroborated by some mitotic and cytokinetic features including development of a microtubular sheath around the prophase nucleus, the almost constant chromosome to pole distance during anaphase, telophase nuclei widely separated by a persistent interzonal spindle, and centripetal plasma membrane invagination. Features, previously unknown in the Charophyceae, include the specific position of the peroxisome lying between the nucleus and adjacent cell wall during interphase and mitosis, the extensive array of microtubules radiating from the centrioles located at the presumptive poles at prophase, involvement of coated vesicles in the furrowing process, and occurrence of transversely aligned cleavage microtubules. Placement of Chlorokybus in the order Klebsormidiales is proposed.     

ULTRASTRUCTURE OF MITOSIS AND CYTOKINESIS IN KLEBSORMIDIUM MUCOSUM NOV. COMB., FORMERLY ULOTHRIX VERRUCOSA (CHLOROPHYTA)1

A transmission electron microscopy study of dividing cells of Ulothrix verrucosa Lokhorst has provided clear evidence that this species differs in many respects from other Ulothrix Kützing species. These differences include the presence of a microtubular sheath around the prophase nucleus, the complete disintegration of the nuclear envelope coinciding with the proliferation of extranuclear microtubules into the prometaphase nucleus and the intrusion of vacuoles into the interzonal spindle region in between the widely separated telophase nuclei. This necessitates the transfer of Ulothrix verrucosa to the charophycean genus Klebsormidium Silva, Mattox and Blackwell. The new combination Klebsormidium mucosum is proposed. On account of its mitotic pattern, this species can be placed in the (charophycean) evolutionary line towards the higher plants. However, because of its cytokinesis (annular centripetal ingrowth of the plasmalemma) this species probably should be considered as a blind offshoot of this line. It is emphasized that furrowing green algae with a persistent interzonal spindle at telophase (including the presently studied alga) often show an ill-defined cytokinetic microtubular system.     

MITOSIS AND CYTOKINESIS IN SESSILE SPORANGIUM OF TRENTEPOHLIA AUREA (CHLOROPHYCEAE)1

An ultrastructure study of mitosis and cytokinesis in the sessile sporangium of Trentepohlia aurea (L.) Mart, was made to clarify the phylogenetic position of the alga. Mitosis was closed and centric at late anphase with cytokinesis involving the production of cleavage membranes by dictyasames between the numerous, well-separated daughter nuclei. Neither phycoplast nor phragmoplast microtubules were observed during cytokinesis. The lack of phycoplast microtubules and the presence of multilayered structures in flagellated cells suggest Trentepohlia is phylogenetically related to those green algae thought to have given rise to the land plants. The primitive type of mitosis and the lack of microbodies suggest that the ancestors of Trentepohlia may have branched off from this line relatively early.     

似金隐藻有丝分裂及胞质分裂的观察

似金隐藻(Cryptomonas chrysoidea)是从青岛附近渤海湾海水中分离得到的一种单细胞藻类。对它的胞质分裂和有丝分裂进行的观察表明,它的胞质分裂在有丝分裂的中期开始,细胞前端沟口处先开始分裂,继而沿纵轴纵沟处形成一收缩沟完成的。似金隐藻的有丝分裂过程中没有染色体和着丝点形成;核膜进入中期时完全消失,纺锤体呈桶状,微管通过染色质团中的通道或直接与染色质团块相联;在后期和末期,两块分开的染色质团十分靠近相应的色素体内质网膜。本文对其分裂过程进行了讨论。     

CELL DIVISION IN THE FILAMENTOUS PLEURASTRUM AND ITS COMPARISON WITH THE UNICELLULAR PLATYMONAS (CHLOROPHYCEAE)1

At prophase in Pleurastrum, extranuclear spindle microtubules develop from the region of centrioles, which lie lateral to the nucleus midway between the future sites of the metaphase spindle poles. The microtubules then move laterally to overarch the nucleus and finally become incorporated into the spindle. The centrioles do not migrate and therefore lie in the same plane as the chromosomes at metaphase. At telophase, 2, more different systems of microtubules develop from the vicinity of the centrioles—a phycoplast and extensive arrays of microtubules that ensheath the daughter nuclei. Cell division in the filamentous Pleurastrum is compared to that in the green flagellate, Platymonas. The similarities between cell division in the 2 algae are interpreted as evidence: (i) that rhizoplasts (which in Platymonas resemble myofibrils) are somehow homologous to microtubules; and, (ii) that cell division in Pleurastrum differs from cell division in other examined filamentous chlorophycean genera because Pleurastrum has an independent evolutionary origin from a monad with Platymonas-like characteristics.     

Analysis of spindle microtubule organization in untreated and taxol-treated PtK1 cells.

Taxol, a microtubule stabilizing agent, has been used to study changes in spindle microtubule organization during mitosis. PtK₁ cells have been treated with 5 μg/ml taxol for brief periods to determine its effect on spindle architecture. During prophase taxol induces microtubules to aggregate, particularly evident in the region between the nucleus and cell periphery. Taxol induces astral microtubule formation in prometaphase and metaphase cells concomitant with a reduction in spindle length. At anaphase taxol induces an increase in length in astral microtubules and reduces microtubule length in the interzone. Taxol-treated telophase cells show a reduction in the rate of furrowing and astral microtubules lack a discrete focus and are arranged more diffusely on the surface of the nuclear envelope. In summary, taxol treatment of cells prior to anaphase produces an increase in astral microtubules, a reduction in kinetochore microtubules and a decrease in spindle length. Brief taxol treatments during anaphase through early G₁ promotes stabilization of microtubules, an increase in the length of astral microtubules and a delayed rate of cytokinesis.     

FINE STRUCTURAL STUDIES ON THE INTERPHASE AND DIVIDING APICAL CELLS OF SPHACELARIA TRIBULOIDES (PHAEOPHYTA)1

The apical cells of Sphacelaria tribuloides Menegh. are larger than other thallus cells, contain more organelles and appear polarized. Their tip portion, where they grow, contains a well developed Golgi apparatus, abundant endoplasmic reticulum (ER) membranes, mitochondria, chloroplasts and a large number of small vacuoles. It seems likely that a continuous flow of membranous material from the ER membranes to the dictyosomes and from the latter to the plasmalemma of the extending tip portion takes place. In contrast, the basal pole possesses fewer organelles and is occupied mainly by large-sized, sometimes central vacuoles. The apical cells undergo two distinct types of highly asymmetrical differential divisions giving rise to cells of the thallus and hair initials. During the early stages of mitosis the nuclear envelope remains intact, except for fenestrated poles. Microtubules pass through the fenestrae into the nucleoplasm. During meta-phase, a typical chromosome plate is organized. The sites of attachment of spindle microtubules to the chromosomes are structurally different from the rest of the chromosomes. At late anaphase, the nuclear envelope breaks down completely. During telophase, a new membrane encloses the chromosomes which are decondensed and the nucleoli are reorganized. Cytokinesis proceeds long after mitosis at a stage in which the nuclei have increased in size and have moved farther apart. A membranous furrow develops centripetally, without the participation of microtubules. However, microtubules traverse the thin cytoplasmic strands which, in both interphase and cytokinetic cells, meander among the vacuoles of the basal pole of the cell and the internuclear space. Dictyosomes appear to be involved in the subsequent wall deposition.     

MITOSIS AND CYTOKINESIS IN ASTEROMONAS GRACILIS, A WALL-LESS GREEN MONAD1

Asteromonas gracilis Artari remains motile throughout cell division. Basal bodies separate and replicate at prophase. They are located lateral to the poles of the closed metaphase spindle. Kinetochores appear at late metaphase. Chromosomes move to the poles and extensions of the nuclear envelope develop into the pyrenoid at anaphase. The interzonal spindle disintegrates at telophase and a diffuse phycoplast is present. Cytokinesis proceeds rapidly from the anterior region of the cell. Newly formed daughter cells have four narrow-banded rootlets and both distal and proximal fibers connect the basal bodies. Features of cell division in Asteromonas are compared to those in other algae, particularly Dunaliella and Chlamydomonas.     

CELL DIVISION AND COLONY FORMATION IN THE GREEN ALGA COELASTRUM (CHLOROCOCCALES)1

Multinucleate cells of Coelastrum undergo precisely directed cytokinesis, guided by phycoplast microtubules, to form a number of uninucleate daughter cells which subsequently adhere to form characteristically patterned aggregates. As there is no movement of the daughter cells relative to one another before their adhesion, the disposition of cells in daughter colonies reflects the pattern of cytokinesis of parent cells. Centrioles lie at the poles of the mitotic nuclei which are partially enclosed by a perinuclear envelope of endoplasmic reticulum. The centrioles disappear at the time of cytokinesis of the parental cell and apparently reform de novo once the daughter cells have acquired a cell wall following their adhesion. The trilaminar layer of cell wall, often termed the pectic layer, does not stain with ruthenium red and resists acetolysis suggesting that it contains sporopollenin rather than pectin.     

红豆杉细胞悬浮培养生产紫杉醇研究进展

介绍了近年来由红豆杉细胞培养生产紫杉醇领域取得的进展,其中特别介绍了由紫杉醇生物合成途径及其代谢酶类的研究发展而来的用基因工程方法改良细胞系,为提高紫杉醇产量而采取添加前体物质,诱导子,抑制子等方面的研究。     

紫杉醇及其衍生物的研究概况与发展趋势

本文分别从生物资源,药理学,化学合成和组织培养方面对红豆杉植物中紫杉醇及其衍生物的研究工作进行了综述,从中可以了解紫杉醇及其衍生物的研究概况与发展趋势。     

PHRAGMOPLASTS IN CYTOKINESIS OF CEPHALEUROS PARASITICUS (CHLOROPHYTA) VEGETATIVE GELLS 1

Cytokinesis in apical cells of actively growing cultures of Cephaleuros parasiticus Karsten sporangiate thalli was examined with transmission electron microscopy. A massive, interzonal cytokinetic microtubule spindle is anchored at its poles to the medial surfaces of the daughter nuclei at telophase. Later, the daughter nuclei are widely separated and no longer associated with the interzonal spindle; however, the spindle retains its shape and becomes a distinct phragmoplast with an array of vesicles, presumably derived from dictyosomes, aligned in the division plane. Fusion of the vesicles gives rise to a thin cell plate. Some bundles of microtubules in the spindle appear to mark the sites of plasmodesmata formation, but no endoptasmic reticulum is directly involved in plasmodesmata formation. No infurrowing or phycoplast array of microtubules is involved in the cytokinesis. The relationship, if any. between the metaphase-anaphase mitotic microtubule system and the interzonal cytokinetic spindle has not been determined. Cephaleuros parasiticus isone of only four green algae now known to contain a higher plant-like phragmoplast and cytokinetic process. The observations reported can be interpreted as very strong evidence for a phylogenetic affinity between the Trentepohliaceae and the Charophyceae, but consideration of ulvophycean features of the Trentepohliaceae such as motile cell ultrastructure and life histories precludes unequivocal assignment of the family to either the Charophyceae or Ulvophyceae.     

MITOSIS IN THE OCTAFLAGELLATE PRASINOPHYTE,PYRAMIMONAS AMYLIFERA (CHLOROPHYTA)1

Cell division is described in the octaflagellate prasinophyte Pyramimonas amylifera Conrad and is compared in related genera. Basal bodies replicate at preprophase and move toward the poles. Cells remain motile throughout division. The nuclear envelope disperses and chromosomes begin to condense at prophase. Pairs of multilayered kinetochores are evident on the chromosomes of the metaphase plate. Spindle microtubules extending from the region of the basal bodies and rhizoplasts attach to the kinetochores or extend from pole to pole. Numerous vesicles and ribosomes have entered the nuclear region and the incipient cleavage furrow invaginates. The chromosomes move toward the poles at anaphase leaving a broad interzonal spindle between the two chromosomal plates. The nuclear envelope reforms first around the chromatin on the side adjacent to the spindle poles and later on the interzonal side. The cleavage furrow progresses into the interzonal spindle at telophase. By late telophase the nucleoli have reformed and the chromosomes have decondensed. The interzonal spindle has not been observed late in telophase. As the cleavage furrow nears completion the cells begin to twist and contort, ultimately separating the two cells.     

稀土化合物对悬浮培养红豆杉细胞紫杉醇生产和释放的影响

采用稀土化合物硝酸镧、硫酸铈铵、硝酸亚铈处理悬浮培养的红豆杉细胞,对八个非外泌型细胞株进行了研究。它们的紫杉醇产量在０．０５￣１５．４４ｍｇ／Ｌ之间,释放率在０％￣２７％之间不同的细胞株对不同稀土化合物的响应是不同的。除ＴＮ、ＮＦ细胞株紫杉醇产量有所下降外,其他细胞株均表现紫杉醇的产量不同程序的提高;Ｅ２细胞株对稀土元素的促渗透作用不敏感。硝酸镧、硫酸铈铵、硝酸亚铈三种稀土化合物中,硝酸亚铈对Ｄ４     

The effect of cytochalasin J on kinetochore structure in PtK1 cells is mitotic cycle dependent

Mitotic PtK1 cells were arrested in mitosis with nocodazole to determine the effect of cytochalasin J (CJ) on kinetochore structure in arrested and nocodazole-released cells. In previous studies it was shown that CJ had a more pronounced effect on alteration of kinetochore structure and spindle microtubule (MT) architecture when applied during prophase or prometaphase. In this study, mitotic cells were treated at preanaphase for 10 min with 1 microg/ml nocodazole, or in 1 microg/ml nocodazole and 10 microg/ml CJ to allow for the advancement of the 'mitotic clock'. Thus it can be determined if either changes in the timing of mitosis, the maturation of the kinetochore, and/or the lack of MT connection to the kinetochore affects the ability of CJ to detach or alter the attachment of chromosomes to the developing spindle. Preanaphase cells treated with 1 microg/ml nocodazole for 10 min and released into 10 microg/ml CJ showed significant changes in MT organization and kinetochore structure. MTs nucleated at the centrosome are fragmented and kinetochore structure was significantly altered showing only two laminae with few MTs inserted into this structure. Preanaphase cells treated with 1 microg/ml nocodazole and 10 microg/ml CJ for 10 min and released into 10 microg/ml CJ showed similar, but more pronounced, effects on kinetochore structure and spindle MT organization. We interpret these results to suggest that CJ treatment has a greater effect on MT attachment and kinetochore structure in nocodazole pre-treated cells, where the kinetochore structure is mature and the mitotic cycle has been advanced.     

磷酸戊糖途径的调节对红豆杉细胞生长及紫杉醇合成的影响

在正常的红豆杉细胞悬浮培养过程,葡萄糖-6-磷酸脱氢酶(G6PDH)活性的变化趋势与生物量的基本相似。而在chitosan处理的细胞中G6PDH活性升高而生物量下降。100 mg·L^-1 chitosan和500mg·L^-1 chitosan均对细胞G6PDH具有诱导作用,且后者的诱导强度较前者的高。乙二醇双2-氨基乙基醚四乙酸(EGTA)的加入降低chitosan对细胞G6PDH的诱导程度,显示chitosan对G6PDH的诱导需要Ca²⁺的参与。谷胱甘肽(GHS)的处理可反馈抑制chitosan对细胞G6PDH的诱导。通过分析调节后G6PDH的各种活性与细胞中紫杉醇产量的关系,认为采用合适的处理方法调节磷酸戊糖途径,有利于红豆杉细胞合成紫杉醇。     

ULTRASTRUCTURE OF CELL DIVISION AND REPRODUCTIVE DIFFERENTIATION OF MALE PLANTS IN THE FLORIDEOPHYCEAE (RHODOPHYTA): CELL DIVISION IN POLYSIPHONIA1

Cell division in the marine red algae Polysiphonia harveyi Bailey and P. denudata (Dillwyn) Kutzing was studied with the electron microscope. Cells comprising the compact spermatangial branches of male plants were used exclusively because of their small size, large numbers and the ease with which the division planes can be predetermined. Some features characterizing mitosis in Polysiphonia confirm earlier electron microscope observations in Membranoptera, the only other florideophycean algae in which mitosis has been studied in detail. Common to both genera are a closed, fenestrated spindle, perinuclear endoplasmic reticulum, a typical metaphase plate arrangement of chromosomes, conspicuous, layered kinetochores, chromosomal and non-chromosomal microtubules, and nucleus associated organelles (NAOs) known as polar rings (PRs) located singly in large ribosome-free zones of exclusion at division poles in late prophase. However, other features, unreported in Membranoptera, were observed consistently in Polysiphonia. These include the presence of PR pairs in interphase-early prophase cells, the attachment of PRs to the nuclear envelope during all mitotic stages, the migration of a single PR to establish the division axis, a prominent, nuclear envelope protrusion (NEP) at both division poles at late prophase, the prometaphase splitting of PRs into proximal and distal portions, and the reformation of post-mitotic nuclei by the separation of an elongated interzonal nuclear midpiece at telophase. During cytokinesis, cleavage furrows impinge upon a central vacuolar region located between the two nuclei and eventually pit connections are formed in a manner basically similar to that reported for other red algae. Diagrammatic sequences of proposed PR behavior during mitosis are presented which can account for events known to occur during cell division in Polysiphonia. Mitosis is compared with that reported in several other lower plants and it is suggested that features of cell division are useful criteria to aid in the assessment of phylogenetic relationships of red algae.     

ULTRASTRUCTURE OF VEGETATIVE AND DIVIDING CELLS OF THE UNICELLULAR RED ALGAE RHODELLA VIOLACEA AND RHODELLA MACULATA1

Rhodella violacea (Kornmann) Wehrmeyer and Rhodella maculata Evans were investigated for ultrastructural details of vegetative and dividing cells. Rhodella violacea has a nuclear projection into the pyrenoid similar to that found in R. maculata, although the nuclear projection in R. maculata traverses a starch-lined area before contacting the pyrenoid. Unlike most, red algae, the two Rhodella species lack a peripheral encircling thylakoid in the chloroplast and have dictyosomes associated solely with endoplasmic reticulum (ER) instead of with both mitochondria and ER. Both species also have a well-developed peripheral system of ER connected to the plasmalemma by tubules, a situation found only in red algal unicells, Cell division was studied primarily in R. violacea; a less thorough examination of R. maculata showed no essential differences. Both have small, double-ringed, nucleus-associated organ files (NAOs) surrounded by moderately electron-dense material, metaphase–anaphase polar gaps in the nuclear envelope, absence of perinuclear ER. and short interzonal spindles. This pattern of mitosis is similar in most respects to that reported in the unicell Flintiella. Following mitosis, microtubules extend from the region of each NAO to its associated nucleus and to the undivided pyrenoid. The NAOs appear to apply tension to the nuclei and the pyrenoid and may be the mechanism for ensuring equal partitioning of both organdies. Two different forms of pyrenoid-nucleus association occur during mitosis. Nuclear projections into the pyrenoid, prevalent during interphase and early stages of mitosis, recede at metaphase. Then, the pyrenoid extends protrusions into the nuclear polar areas, forming a cup that partially surrounds the nucleus. Cell division and vegetative characters confirm the close taxonomic affinity of these two species of Rhodella and support their separation from the genus Porphyridium.     

Taxol对培养的人成纤维细胞内微管组装的影响

本实验用微管的PAP免疫酶细胞化学方法,研究了培养的小儿包皮成纤维细胞及其分离的中心体在taxol的作用下对微管组装的影响。实验结果表明taxol对低温(4℃)和微管解聚药物的处理具有拮抗作用,它阻止微管解聚,对微管具有稳定作用,并观察到taxol可降低中心体对微管组装所需的管蛋白临界浓度,增强中心体对微管的组装能力。Taxol对细胞内微管的影响,主要表现在促使微管呈束状浓集化,并随处理时间的延长,这种浓集化表现愈益明显,导致破坏胞质CMTC的正常分布。由于taxol能使微管浓集化,抑制其解聚,使得细胞从G_2期进入M期后,微管不解聚,从而不能形成正常的纺锤体,胞质不分裂,最后导致细胞微核化。用秋水仙酰胺处理后再加taxol时,我们观察到细胞CMTC与正常未经处理的细胞CMTC比较,呈相反的分布现象,这可能与秋水仙酰胺促使中心体与细胞核分离和taxol增强中心体对微管的组装有关。     

紫杉醇在氧化铝层析过程中的稳定性研究

本文比杉醇在氧化铝层析分离紫杉烷过程中的稳定性进行了实验分析。结果表明,紫杉醇在柱中的仪时间、流动相中甲醇浓度、介质的含水量以及操作温度是影响稳定性的主要因素。当停留时间控制在４５ｍｉｎ以内,氧化是质经过脱水处理,降低甲醇浓度及温度控制在２５℃以内时,可减少紫杉醇在氧化铝层析过程中的降解。