Ultrastructure ofCatharanthus roseus cells culturedin vitro and exposed to conditions for alkaloid accumulation

Summary Periwinkle (Catharanthus roseus) cells cultured in 1-B 5 medium display the ultrastructure of parenchyma cells. The parenchyma character remained unchanged when cells were exposed to any one of three different conditions effecting alkaloid accumulation. Transfer of cells to alkaloid production medium for 2 weeks (condition 1) accorded two special features,i.e., unusually big lipid droplets in the cytoplasm and, upon fixation, one or several electron-dense droplets of spongy precipitate in vacuoles. Among hormone-autotrophic cultures (condition 2) some cells showed a fine electron-dense vacuolar precipitate. Addition ofPhythium homogenate (fungal elicitor) to cells cultured in 1-B 5-medium for 10 days (condition 3), cells showed a frequent appearance of singular big lipid droplets in the cytoplasm, whereas vacuoles remained devoid of precipitate. The appearance of big lipid droplets and of vacuolar precipitate is interpreted as progressing cytodifferentiation, but is coincidental with alkaloid accumulation.NRCC no. 24524.     

紫萁孢子囊发育中多糖和脂滴的细胞化学观察

采用光镜、透射电镜和细胞化学技术,对紫萁孢子囊发育过程中孢壁的超微结构和孢子囊内多糖和脂滴的分布及其动态变化进行研究,以探讨紫萁孢子囊发育过程中多糖和脂滴的代谢特征,为蕨类孢子发生的研究提供基础资料。结果表明:(1)紫萁孢子囊由1层囊壁细胞、2层绒毡层和产孢组织构成。(2)紫萁孢子壁由发达而分2层的外壁(外壁内层和外壁外层)和薄的不连续的周壁构成,由外壁形成棒状纹饰的轮廓;孢子外壁内层由多糖类物质构成,外壁外层和周壁均含有脂类物质。(3)在紫萁孢原细胞中观察到少量脂滴;随着紫萁孢壁的形成,囊壁细胞中淀粉粒的大小逐渐变小、数目先增加后减少,它们转运到内层绒毡层原生质团并转化为孢粉素前体物质,再穿过原生质团内膜表面进入囊腔,成为孢粉素团块或以小球形式填加到孢子表面形成孢壁。(4)紫萁孢子囊将多糖类营养物质转化为脂类,以脂滴的形式储藏在孢子中。     

Zygote formation inCosmarium botrytis studied by scanning and transmission electron microscopy,phase-contrast,and Calcofluor staining

Summary Rapid zygote formation byCosmarium botrytis was induced in a liquid medium by incubation in 5% CO₂. Conjugation and zygote formation were studied by SEM, TEM, phase-contrast, and Calcofluor fluorescence microscopy. It was observed that the cells divided immediately prior to conjugation and formed Calcofluor fluorescent conjugation papillae as soon as the primary wall was shed. The conjugating cells and the resultant zygote were envelopped by a non-fluorescent mucilagenous envelope which was eventually pierced by the zygote spines, but never shed. The very young smooth-walled zygote had a thick Calcofluor fluorescent wall. At that stage the zygote could be plasmolysed in 0.4 M mannitol, but no protoplast could be induced to emerge even with the addition of up to 5% Cellulysin; probably indicating that the zygote wall composition and structure is different from that of the secondary wall of the vegetative cells, particularly in the absence of mucilage pores.     

Exceptional long-term starvation ability and sites of lipid storage of the Arctic pteropod <Emphasis Type="Italic">Clione limacina</Emphasis>

The Arctic pteropod Clione limacina was collected in Kongsfjorden, Svalbard, in mid June 2004, to study the lipid metabolism within the sites of lipid storage structures during long-term starvation. Animals survived in an aquarium without any food for nearly 1 year (356 days). Size, number of lipid droplets, dry and lipid mass, lipid class and fatty acid compositions of C. limacina were determined and separately analysed for the digestive gland and the remaining integument. During the starvation period, animals shrunk from 22.4 to 12 mm in length on average, and the number of lipid droplets decreased from 1,600 to 1,000 per animal. Dry mass (DM) and total lipid mass both dropped by about 80% from day 200 to the end. The lipid content as percentage DM of the total organism did not decrease significantly ranging from 43.8 to 32.3%DM. The lipid content of the trunk was moderate with about 20%DM. The digestive gland was very rich in lipids with more than 70%DM throughout the experiment and is the major site of lipid metabolism and storage. Triacylglycerols (TAG) decreased, in the total organism, from high initial levels of 62.6 to 43% of total lipid at the end. In contrast, the proportions of 1-O-alkyldiacylglycerols [diacylglycerol ethers (DAGE)] remained almost constant, varying between 20.4 and 28.4%. In the digestive gland, TAG ranged from 60.3 to 64.8% and DAGE from 23.6 to 32.2% from day 200 to the end of the experiment. TAG and DAGE of the trunk were most likely located in the lipid droplets and were almost depleted at the end of starvation. Besides their function as lipid deposit DAGE may also act as protecting substance against bacterial and fungal infections. During the first 200 days of starvation, the fatty acid compositions showed only small variations. Thereafter, fatty acids typical for storage lipids decreased in all body compartments. In adaptation to long periods of food scarcity, C. limacina has evolved various strategies as body shrinkage, utilisation of body constituents not essential for survival, a very low metabolism and slow lipid consumption.     

银杏套细胞发育的解剖学研究

银杏套细胞起源于近雄配子体表达的颈卵器母细胞周围细胞,中央细胞形成时,套细胞呈明显的一层,围绕颈卵器紧密排列,随着颈卵器的发育,套细胞体积逐渐地大,细胞质变浓厚,细胞质中脂滴增多,套细胞和中央细胞的接触壁开始出现局部加厚,在颈卵器发育的泡沫化阶段,套细胞和中央细胞的接触壁不均匀加厚较为显著,在较薄的区域可见胞间连丝,受精前,套细胞中液泡增多,脂滴迅速减少,造淀粉体增大,套细胞与卵细胞的接触壁的不均匀加厚非常明显,精核进入颈卵器以后,卵细胞与套细胞的接触壁和卵细胞的质膜之间形成一个薄厚不均的间隔层,受精卵分裂时,受精卵细胞与套细胞接触壁的凹陷处可见许多小泡和内质网,游离核期时,套细胞内出现大量小液泡,细胞内含物迅速消失,套细胞外形变长,胚胎长出颈卵器后,套细胞逐渐解体消失。     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.     

Fine structure of the corpus allatum of the female blow-fly Calliphora erythrocephala

Summary An electron microscopical study of the corpus allatum (CA) of the adult female Calliphora was undertaken.The cells have a very irregular shape. Light and dark cells are found. Mitochondria occur in great numbers. Microtubules are frequently observed. Free ribosomes are plenty, but rough-surfaced reticulum is scarce. Golgi complexes are not very conspicuous. Axons, mostly containing neurosecretory granules, are frequently found between the cells.The active corpus allatum is remarkable by the numerous lipid droplets and the abundance of tubular agranular reticulum. The reticulum sometimes forms aggregates from which vacuoles are budded off. The vacuoles lose their membrane, at the same time becoming slightly electron opaque, thus being transformed into lipid droplets.It is tentatively postulated that the hormone (or a precursor) is synthesized in the tubules of the agranular reticulum, collected in the vacuoles, and, when the membrane disintegrates, it is dissolved in lipid. The lipid droplets are thought to be released into the haemolymph through the surface of the gland or via intercellular channels.The inactive corpus allatum of the six days old sugar fed flies is small and more or less shrunken. The agranular reticulum is poorly developed, vacuoles are small, and lipid droplets few. The reticulum tends to form whorls, which eventually may possibly be transformed into myelin figures.We wish to express our gratitude to the Danish Natural Science Research Council for placing a Zeiss electron microscope at our disposal, and to the Carlsberg Foundation for supporting our work with grants. We are grateful to Prof. C. Overgaard Nielsen for laboratory facilities, and we are indebted to Mrs. Eva Jensen for her skilful technical assistance.     

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Supported by National Natural Science Foundation of CHINA (30170060)     

SEXUAL REPRODUCTION OF PERIDINIUM WILLEI (DINOPHYCEAE)1

Sexual reproduction was induced in the dinoflagellate Peridinium willei Huitfeld-Kass when exponentially growing cells were inoculated into nitrogen deficient medium. Small, naked vegetative cells produced by division of thecate cells acted as gametes. The zygote remained motile 13–14 days, during which time it enlarged and the theca formed became warty. Fourteen to 15 days following plasmogamy the zygote was nonmotile with the protoplast contracted. A large red oil droplet appeared and the wall thickened, becoming chitinized. Hypnozygotes with 4 nuclei were observed 7–8 wk following formation. Meiosis was inferred. The hypnozygote germinated, within 8 wk producing one post-zygotic cell retaining the red oil droplet. This cell divided within 24 h into 2 daughter cells each with a prominent red oil droplet. These daughter cells divided after 2 to 3 days into ordinary vegetative cells. Attempts to induce sexual reproduction by inoculation of cells into media deficient in a number of basic elements were unsuccessful.     

Zygote Formation in Blakeslea trispora: Morphological Peculiarities and Relationship with Carotenoid Synthesis

Changes associated with zygospore formation in the mucorous fungus Blakeslea trispora were studied. Zygospores are dormant cells with thickened cell walls and large central lipid vacuoles containing large amounts of lycopene. We established for the first time that B. trispora gametangia of different sexes differ in their carotenoid content and revealed that zygote formation involves a novel structure that consists of densely intertwined hyphae. Using inhibitory analysis (blocking -carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene producion results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on -carotene synthesis.     

CONJUGATION IN SPIROGYRA1

Conjugating filaments of Spirogyra were examined with both light and electron microscopes. Initially 2 or more filaments of Spirogyra were attached by mucilagenous material. Papillae appeared first in one filament and then in adjacent positions on the other filament. Subsequent growth of papillae separated the conjugating filaments; wall microtubules disappeared in papillae as they elongated. Golgi activity then increased markedly only in the male filament; mucilage production by these Golgi coincided with contraction of the male gamete from its cell wall and may be responsible for its subsequent migration. The end walls separating papillae dissolved to form the conjugation tube, allowing gamete union. The male protoplast then migrated through the tube and further cytoplasmic condensation formed an elliptical-shaped zygote. During the migration phase, zygote wall formation was initiated and numerous active Golgi apparently contributed material to it. Early zygote maturation was characterized by rapid wall formation and an increase in lipid droplets.     

Changes of the cytoplasmic ultrastructure during development of sclerotia in Claviceps purpurea (Fr.) Tul.

The development of sclerotia of Claviceps purpurea was investigated by light and electron microscopy. During the first days after infection sterigma and conidiospores are formed. The spores show a moderately developed vacuolar system, they are thick walled and contain about 20% lipid (related to the cell volume) embedded in glycogen. The sterigma are cylindrical unicellular hyphae with electron dense cytoplasm and isolated strongly contrasted lipid droplets. In maturing sclerotia the hyphae become septated with increasingly thick cell walls and a large lipid content. The lipid forms small droplets in young cells, while in the mature sclerotium it occurs in the form of very large drops, occupying the major part of the cell. Simultaneously the composition of the lipid is changed. The mature cells have several nuclei. They are partially connected by osmiophilic substances, forming a network of intercellular spaces.Abbreviations HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - DMSO Dimethylsulfoxide     

Transfer cells and lipid droplets of someValerianaceae

Development, structure and the axial distribution of transfer cells and their lignification were investigated inValerianella locusta, Valeriana officinalis, andV. tuberosa (Valerianaceae). Fundamental new results are: (1) Transfer cells often contain numerous lipid droplets. Within the stem the distribution of cells containing lipid droplets correlates to that of transfer cells. (2) InValeriana officinalis persisting protuberances are frequently found on pit membranes of xylem transfer cells. Lignified transfer cells can undergo a second modification: a layer covering the secondary wall forms wall ingrowths similar to those of transfer cells. (3) Peripheral pith cells, abuting transfer cells, are able to modify into transfer cells. Cambial derivatives are only temporarily developed as transfer cells. (4) Phloem transfer cells are found in vascular bundles of the whole axis. (5) In roots, xylem transfer cells are poorly developed or absent. (6) Oil cells with oil bodies are present in the rape ofValeriana tuberosa. They are absent however in the stem of the species investigated. (7) Tannins occur in elements of the primary cortex, phloem and secondary xylem ofValeriana officinalis.     

多花地宝兰胚囊和胚发育的细胞学研究

为探讨多花地宝兰(Geodorum recurvum)胚胎发育的系统分类学意义,采用石蜡制片法对多花地宝兰胚囊和胚的发育进行解剖学观察。结果表明,在开花前,多花地宝兰胚珠原基发育缓慢,开花授粉后胚珠原基快速发育成"树状二杈分枝结构",随后在"分枝结构"末端形成孢原细胞,开始胚囊发育。多花地宝兰的胚囊发育属于单孢蓼型胚囊,胚珠具有双层珠被。孢原细胞形成后,经过细胞膨大延长发育形成胚囊母细胞,胚囊母细胞经过减数分裂形成线性四分体,在珠孔端形成1个功能大孢子,功能大孢子经过3次有丝分裂形成8核胚囊。多花地宝兰的胚发育具有藜型和紫苑型两种方式。双受精完成后,多花地宝兰合子进行一次橫裂后形成基细胞和顶细胞;基细胞经过多次分裂形成细胞团,细胞团中的细胞向不同方向膨大延长形成多个胚柄细胞;顶细胞有两种分裂方式,一种是横裂形成藜型胚,一种是纵裂形成紫苑型胚。因此,推测多花地宝兰在兰科植物系统分类学上属于较为原始种。     

耻垢分枝杆菌感染人脐带间充质干细胞模型的建立及其特征

【背景】结核分枝杆菌(Mycobacterium tuberculosis, Mtb)休眠菌形成被认为是潜伏结核感染(latent tuberculosis infection, LTBI)的主要原因,但目前缺乏体内和体外模型进行机制研究。新近研究表明Mtb可感染间充质干细胞(mesenchymal stem cells, MSC)并以休眠状态在细胞中长期存活。然而Mtb感染细胞模型存在周期长和生物安全要求高等问题,需要探索可用的MSC感染细胞模型用于Mtb休眠机制的研究。【目的】建立快速生长型耻垢分枝杆菌(Mycobacteriumsmegmatis,Ms)感染人脐带间充质干细胞(human umbilical cord MSC, hUCMSC)的细胞模型并研究其特征。【方法】取分离好的hUCMSC,流式细胞术鉴定其表面标志性抗原;以Ms菌株感染hUCMSC,DiI标记细胞膜,荧光显微镜下观察细胞吞噬作用;平板法计数Ms胞内存活率;油红O染色观察细胞脂滴形成;鬼笔环肽荧光染色观察细胞骨架变化;实时荧光定量PCR(realtimequantitativePCR,RT-qPCR)检测Ms...     

Occurrence and metabolic significance of microbodies in trophic hyphae of the nematophagous fungus Arthrobotrys oligospora

This paper describes the results of an ultrastructural study on the subcellular events occurring in nematode-infecting (trophic) hyphae of the nematophagous fungus Arthrobotrys oligospora. In early stages of the infection process (30 min-4 h), the infection bulb and developing trophic hyphae are characterized by a highly proliferated endoplasmic reticulum (ER). Its membranes often appeared vesiculated and occur in close association with the cell membrane of the cells. Upon further invasion of the nematode, lipid droplets developed in the trophic hyphae; these droplets were first observed 4–5 h after the infection but were abundantly present after 24–36 h. Along with the formation of lipid droplets proliferation of microbodies was observed. These organeles were characterized by the presence of catalase and thiolase and were frequently observed in close association with the lipid droplets. Later on the lipid droplets disappeared. During this period new vegetative mycelium developed from the trap that had originally captured the nematode. Our results suggest that part of the nutrients released from the nematode are first converted into lipids by the fungus which in turn are degraded via the -oxidation pathway and further metabolized to support growth of new vegetative hyphae.     

Cytological identification of cell types in the testis of Esox lucius and E. niger

Summary Testes of Esox lucius and Esox niger were investigated histologically, cytochemically, and ultrastructurally in reproductive fish. Intralobular Sertoli cells possessed numerous lipid droplets in Esox lucius, but not in Esox niger. In both species, interlobular cell types included myoid cells and lipid-negative Leydig cells within the extravascular space. Evidence is presented for a contractile network of myoid cells within the testes of these teleosts. The presence of Leydig cells and myoid boundary cells in the testis of Esox lucius refutes the reported homology between lobule boundary cells and Leydig cells in this species.     

Development and cellular organization ofPinus sylvesfris pollen tubes

The organization ofPinus sylvestris pollen tubes during growth was studied by video microscopy of living cells and by electron microscopy after freeze-fixation and freeze-substitution (FF-FS). Pollen germinated and the tubes grew slowly for a total period of about 7 days. Some of the grains formed two tubes, while 10–50% of the tubes ramified. These features are in accordance with development in vivo. The cytoplasmic hyaline cap at the tip disappeared during the 2nd or 3rd day of culture. Aggregates of starch grains progressively migrated from the grain into the tube and later into the branches. Vacuoles first appeared at day 2 and eventually filled large parts of the tube. The tube nucleus was located at variable distances from the tip. Some of the organelles showed linear movements in a mostly circulatory pattern, but the majority of the organelles showed brownian-like movements. Rhodamine-phalloidin-stained actin filaments had a gross axial orientation and were found throughout the tube including at the tip. The ultrastructure of pollen tubes was well preserved after FF-FS, but signs of shrinkage were visible. The secretory vesicles in growing tips were not organized in a vesicle cone, and coated pits had a low density with only local accumulations, which is in accordance with slow growth. The mitochondria contained small cristae and a darkly stained matrix and were located more towards the periphery of the tube, indicating low respiratory activity and low oxygen levels. The dictyosomes carried typical trans-Golgi networks, but some contained less than the normal number of cisternae. Other elements of the cytoplasm were irregularly spaced rough endoplasmic reticulum, many multivesicular bodies, lipid droplets and two types of vacuoles. The typical organization associated with tip growth in angiosperm pollen tubes, e.g.Nicotiana tabacum, was not present inP. sylvestris pollen tubes. The different morphology may relate to the growth rate and not to the type of growth.     

SEXUAL REPRODUCTION IN THE FRESHWATER DINOFLAGELLATE GLOEODINIUM MONTANUM1

The sexual life cycle of Gloeodinium montanum Klebs was examined with light and scanning electron microscopy. In unialgal cultures G. montanum divided predominantly by simple division, giving rise to two nonmotile cells. When placed in fresh medium, 2–4 biflagellated swarmers were formed from the vegetative cells. Swarmers developed directly into vegetative cells or acted as gametes. Both isogamy and anisogamy were observed. Gloeodinium montanum is homothallic. Fusion occurred in the non-motile state producing a large aplanozygote, which germinated after approximately two months to a year or more. Zygote germination liberated four aplanospores. Budding of the zygote, resulting from unequal division of the protoplast and multiple fusion attempts also were observed.     

Stellate cells storing retinol in the liver of adult lamprey,Lampetra japonica

Summary Distribution, localization and fine structure of the stellate cells in the liver of lamprey, Lampetra japonica, were studied during the spawning migration by use of Kupffer's gold-chloride method, fluorescence microscopy for vitamin A (retinol) and electron microscopy. The stellate cells in the lamprey liver differ in some of their properties from those in mammalian livers. Stellate cells which store abundant retinol in lipid droplets, occur not only in the hepatic parenchyma, but also in the dense perivascular and capsular connective tissue of the liver and in the interstitium of pancreatic tissue. In the hepatic parenchyma these cells are located perisinusoidally or along thick bundles of collagen fibrils. The stellate cells display a number of large retinol-containing lipid droplets, granular endoplasmic reticulum, tubular structures, dense bodies, Golgi complex, microtubules, and microfilaments. In the space of Disse, the stellate cells and extracellular fibrilar components such as collagen fibrils and microfibrils (11–12 nm in diameter) are intervened between the two layers of basal laminae. Differentiation and possible functions of the stellate cells in the lamprey liver are discussed.