Location of sites of dopamine storage in megakaryocytes by autoradiography

We have used quantitative electron microscope autoradiography to study the subcellular sites of 3H-dopamine uptake in mouse megakaryocytes after a single intraperitoneal injection. Autoradiographic grains were found to be associated almost exclusively with the vesicles of precursors of monoamine-storage organelles. The labeling intensity (radioactivity) of the demarcation membrane system which is continuous with the plasmalemma was also significantly greater than would be expected. On the other hand, radioactivity associated with the remaining sites in the cytoplasm was not significantly different from that expected of a random distribution. In order to compare 3H-dopamine uptake during cell maturation, light microscope autoradiographic studies were also done. Immature megakaryocytes were labeled slightly, but the number of silver grains increased significantly in maturing cells. Mature megakaryocytes were 2.7 times more radioactive than the maturing cells. Our results suggest that the megakaryocytes were able to accumulate dopamine from the early stages of cell maturation and to store dopamine in precursors of monoamine-storage organelles.     

Occurrence of uranaffin-positive synaptic vesicles in both adrenergic and non-adrenergic nerves of the rat anococcygeus muscle

Summary The uranaffin reaction in rat anococcygeus muscle, which receives a dual innervation of both adrenergic and non-cholinergic, non-adrenergic nerves was examined. Dense reaction product was observed in the vesicular membranes and/or the cores of some synaptic vesicles in the adrenergic nerve terminals. Occasional vesicles were filled up with dense reaction product. In the prominent population of small clear vesicles, however, no dense reaction product was observed. The number of small granular vesicles in the adrenergic nerve terminals was markedly increased after the administration of 5-hydroxydopamine (5-OHDA). These granular vesicles were moderately stained with uranaffin deposit on the cores but their limiting membranes possessed no uranaffin deposit at all.In the non-adrenergic nerve terminals, on the other hand, uranaffin deposit of variable density was observed on the cores of large granular vesicles but never on their limiting membranes or on the small clear vesicles. There was no change in the axon profiles after the administration of 5-OHDA.The possible occurrence of purines in the cores of large granular vesicles in the non-adrenergic nerves is discussed.     

Uranaffin reaction: a new cytochemical technique for the localization of adenine nucleotides in organelles storing biogenic amines.

Amine storage organelles of aldehyde-fixed rabbit platelets have a strong affinity for uranyl ions before their dehydration and appear highly electron-dense when examined by electron microscopy; both the matrix and membrane of these organelles are intensely stained. This affinity, which is also shown by platelets of other species, including healthy reserpinized platelets, which contain no cytochemically demonstrable amine, show a positive uranaffin reaction. However, platelets and megakaryocytes of strains with storage pool deficiency (low adenosine-5'-triphosphate), including patients with Hermansky-Pudlak syndrome, are uranaffin negative. The cytochemical reaction, probably the result of an interaction between UO2++ ions and phosphate groups of 5'-phosphonucleotides, is also observed in adrenal medulla, sympathetic nerve terminals and ganglion cells, suggesting that the technique will be of considerable value in identification of aminergic neurons and in further elucidation of amine storage mechanisms.     

Interaction of lipid bodies with other cell organelles in the maturing pollen of Magnolia × soulangeana (Magnoliaceae)

The pollen grain maturation in Magnolia × soulangeana was studied ultrastructurally and cytochemically using both the light and transmission electron microscope. Emphasis was given on the storage lipid bodies of the vegetative cell (VC) and their interaction with other cell organelles. Stereological analysis of electron micrographs was performed to evaluate the variation in volume density (V_V), surface density, and surface-to-volume ratio (S/V) of various cell organelles during pollen maturation. The size and numerical density of the lipid bodies, and their frequency of association with other cell organelles, were also determined. It was noted that during pollen ontogeny and maturation, the lipid bodies changed their pattern of distribution in the VC cytoplasm, which may be a good marker for the succeeding stages of pollen development. Also, the size, osmiophily, and V_V of the lipid bodies were progressively reduced during pollen maturation whereas the S/V was significantly increased. This seems to indicate that the lipid bodies are mobilized in part during this period of pollen maturation. In particular, the intermediate and mature pollen showed a high percentage of lipid bodies establishing a physical contact with either glyoxysomes, either protein storage vacuoles, or small vesicles presumably originated from dictyosomes. This physical contact was found in both the chemically fixed and rapid freeze-fixed pollen indicating that it is neither artifactual nor casual. On the basis of this intimate association with other cell organelles and the morphometric analysis performed, we suggest that the mobilization of lipid bodies is likely mediated not only by glyoxysomes but also by other catabolic pathways involving the interaction of lipid bodies with either protein storage vacuoles or small Golgi vesicles.     

Chromaffinity, uranaffinity and argentaffinity of small granule-containing (SGC) cells in rat superior cervical ganglia.

A systemic examination on the small granule-containing (SGC) cells in rat superior cervical ganglia was conducted by conventional and cytochemical electron microscopy including chromaffin, argentaffin and uranaffin reactions. According to the fine structure of dense cored vesicles (DCVs) in the cytoplasm, three types of small granule-containing (SGC) cells were revealed--Type I: 90-160 nm vesicles with cores of moderate or low electron density; Type II: 130-330 nm vesicles, polymorphic with highly electron dense cores; Type III: elongated vesicles (170 nm x 60 nm) with cores of moderate to low electron density. The majority of SGC cells were the Type I cells (78%) and Type II and III cells made up 13% and 9% of SGC cell population, respectively. Cytochemical results demonstrated that only the Type II cells displayed a positive chromaffin reaction and all three types of SGC cells showed argentaffinity and uranaffinity. The present study is the first to demonstrate the argentaffin reaction at ultrastructural level in SGC cells of sympathetic ganglia. Based on the results of the present study we also concluded that (1) the DCVs of Type II SGC cells contained noradrenaline and (2) biogenic amines and nucleotides (ATPs) coexisted in the DCVs of all three types of SGC cells.     

Effects of gangliosides on cell maturation of murine megakaryocytes in a liquid culture system

Formation of platelet-producing megakaryocytes, the cytoplasm of which showed the terminal stage of cell maturation, heavy granulation and platelet-fields delineated with demarcation membranes, was observed in a short-term culture system, using megakaryocyte-enriched bone marrow cell suspension. Approximately 6-8% of the megakaryocytes changed to the platelet-producing megakaryocytes during 12-hour incubation. In the presence of inhibitors of energy metabolism, formation of the platelet-producing megakaryocytes was inhibited, suggesting that the process is dependent on energy producing systems. Ganglioside GD1a increased both the number of total megakaryocytes and the ratio of the platelet-producing megakaryocytes to total megakaryocytes, while GM1 did not influence the number of total megakaryocytes, but increased the ratio. Gangliosides GM2, GM3 and GD1b showed little effect on either the number of total megakaryocytes or the ratio. The results suggest that ganglioside GD1a stimulates at least two steps of megakaryocyte maturation, the change of megakaryocytic progenitors to megakaryocytes and the subsequent maturation of megakaryocytes to the platelet-producing megakaryocytes, while GM1 stimulates only the latter step of the maturation.     

Thorotrast uptake and transit in embryonic glia, heart fibroblasts and neurons in vitro

Thorotrast (colloidal ThO₂) is incorporated into coated vesicles, various agranular vesicles and sacs, and a surface-associated system of membranous channels in times as short as 1 min by single cultured glial and heart cells. Thorotrast appears in ‘C’-shaped bodies and in small, dense bodies of the lysosomal series within ca. 25 min. With longer chase periods, thorotrast ‘clears’ from all cytoplasmic organelles except the lysosomal series. The technique of applying thorotrast and using varying chase periods fails to distinguish a class of membranous organelles, located close to the cell periphery, that might serve as a source of new cell surface during locomotory activity. Similarly, thorotrast (colloidal ThO₂) is incorporated into almost all classes of membrane-bounded organelles of growth cones and axons of single nerve cells in vitro in times as short as 1 min. This includes elements of the smooth endoplasmic reticulum. No thorotrast enters the lysosomal granules in this short time. During various chase periods, the tracer disappears from the initial sites of incorporation and accumulates in dense bodies of the lysosome series within growth cones and axons. ‘C’-shaped bodies may be an intermediate in that process. No unique sites of endocytotic activity or of a complete absence of endocytosis were observed that could be correlated with growth cone function and axonal elongation, though the presence of the tracer in agranular sacs of the smooth endoplasmic reticulum in growth cones could reflect hypothesized cycling of cell surface (Bray, 1973).     

毛竹茎纤维次生壁形成过程的超微结构观察

利用透射电镜观察了毛竹（Ｐｈｙｌｌｏｓｔａｃｈｙｓ ｐｕｂｅｓｃｅｎｓ Ｍａｚｅｌ）茎纤维发育过程中次生壁的形成过程。纤维发育早期,细胞具有较大的细胞核和核仁;细胞质浓稠,具有核糖体、线粒体和高尔基体等细胞器。随着纤维次生壁的形成,细胞壁加厚,细胞质变得稀薄,内质网和高尔基体的数量明显增加,并且两者共同参与了运输小泡的形成;在质膜内侧可观察到大量周质微管分布。随着次生壁的进一步加厚及木质化,细胞壁     

Ultrastructural Study of Secondary Wall Formation in the Stem Fiber of Phyllostachys pubescens

Ultrastructural changes in secondary wall formation of Phyllostachys pubescens Mazel fiber were investigated with transmission electron microscopy. Fiber developed initially with the elongation of cells containing ribosomes, mitochondria and Golgi bodies in the dense cytoplasm. During the wall thickening, the number of rough endoplasmic reticulum and Golgi bodies increased apparently. There were two kinds of Golgi vesicles, together with the ones from endoplasmic reticulum formed transport vesicles. Many microtubules were arranged parallel to the long axis of the cell adjacent to the plasmalemma. Along with the further development of fiber, polylamellate structure of the secondary wall appeared, with concurrent agglutination of chromatin in the nucleus, swelling and disintegration of organelles, while cortical microtubules were still arranged neatly against the inner side of plasmalemma. Lomasomes could be observed between the wall and plasmalemma. The results indicated that the organelles, such as Golgi bodies together with small vesicles, rough endoplasmic reticulum and lomasomes, played the key role in the thickening and lignification of the secondary wall of bamboo fiber, though cortical microtubules were correlative with the process as well.     

Secretory organelle biogenesis: Trichocyst formation in a ciliated protozoan

Summary— By classical electron microscopy and immunoelectron microscopy, the biogenesis of trichocyst secretory granules has been followed in the ciliated protozoan Pseudomicrothorax dubius. The very early pre-trichocysts form by fusion of bristle-coated, electron-dense vesicles (dense vesicles) with electron-translucent vesicles (clear vesicles), both of which originate in a well-developed trans-Golgi network (TGN). The pre-trichocyst grows by further fusion with dense and clear vesicles as well as with other pre-trichocysts until it reaches its maximum diameter of about 2 μm. Dense and clear vesicle formation from the TGN has been followed, and the fusion sequence of dense vesicles with the pre-trichocyst has been documented. The contents of the dense vesicles are the precursors of the trichocyst tip, which is composed of four arm-like rods, whereas the shaft precursors are supplied by the clear vesicles. The first evidence of trichocyst shaft formation is the appearance of a paracrystalline, dense core condensation center in the pre-trichocyst. Following shaft formation, the trichocyst tip forms by fusion and condensation of the dense arm precursors along each of the four sides of the shaft. Docking of the fully formed trichocyst in the cell cortex is described. Pre-trichocyst biogenesis in cells grown with and without Se is compared.     

Light and electron microscopic cytochemistry of glycoconjugates in the rectosigmoid colonic epithelium of the mouse and rat

The several cell types in mouse and rat rectosigmoid colon have been examined with light and electron microscopic methods for localizing and characterizing complex carbohydrates. Mucous cells, also termed vacuolated cells, and goblet cells comprised most of the deep crypt epithelium in both species, and absorptive columnar cells and goblet cells mainly populated the more superficial epithelium of the upper crypts and main lumen. Occasional tuft cells and enteroendocrine cells were also encountered. Transitional cells structurally intermediate between mucous cells and absorptive cells contained granules characteristic of mucous cells and vesicles like those of columnar absorptive cells. These intermediate cells supported the concept of replacement of mucous by absorptive cells through transformation of mucous into absorptive cells. The intermediate cells also contained numerous lysosomes often in apparent fusion with mucous granules, indicating crinophagic disposal of mucous granules as a mechanism in the cell transformation. Glycoconjugate in absorptive cell vesicles resembled that coating the apical plasmalemma and appeared to represent the source of the glycocalyx of the brush border. Complex carbohydrate in these vesicles differed cytochemically from that of the mucous cell granules, which release their content into the crypt lumen. The absorptive cell vesicles, therefore, constitute an organelle distinct from the mucous cell granules rather than an atrophic form of the latter in a more mature cell. Goblet cells differed in failing to transform morphologically with age but changed in the cytochemical characteristic of their secretion during migration up the crypts. Terminal N-acetylglucosamine residues diminished, while terminal sialic acid-galactose dimers increased during the upward migration, indicating activation of glycosyl transferase synthesis in relation to goblet cell maturation. Glycoconjugate in secretion of mucous cell granules differed markedly from that in goblet cell granules, and content of both organelles differed from that of absorptive cell vesicles. However, secretion in mucous cell granules appeared generally similar for mice and rats with minor exceptions, and secretion in goblets of mice generally resembled that in goblets of rats. Cells interpreted tentatively as Kulchitsky cells stained for high content of fucose with the Ulex europeus I lectin. Globoid leukocytes infiltrating the epithelium of the rat but not the mouse rectosigmoid colon resembled globoid leukocytes in rat tracheal epithelium and, like the latter, appeared to derive from mast cells.     

Novel fluorescent dyes based on oligopropylamines for the in vivo staining of eukaryotic unicellular algae

Weakly basic fluorescent dyes are used to visualize organelles within live cells due to their affinity to acidic subcellular organelles. In particular, they are used to stain the silica deposited in the silica deposition vesicles (SDVs) of diatoms during the course of their frustule synthesis. This study involved the synthesis of fluorescent dyes derived from oligopropylamines, compounds similar to those found in diatoms. The dyes were obtained by reacting oligopropylamines with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The reaction was realized using methylated oligopropylamines with two or three nitrogen atoms and yielded two novel fluorescent dyes: NBD-N2 and NBD-N3. The dyes appeared to be highly efficient in the in vivo staining of growing siliceous frustules of diatoms at concentrations at least 10 times lower than those required for staining with HCK-123. NBD-N3 also efficiently stained other subcellular vesicles of eukaryotic unicellular algae. NBD-N2 stained only growing diatom frustules, whereas NBD-N3 also stained various subcellular organelles of different eukaryotic unicellular algae. NBD-N2 and NBD-N3 were not removed from stained diatom frustules by drastic treatments with H₂SO₄ and H₂O₂. Fluorescent silica can also be obtained by its chemical precipitation in the presence of NBD-N2 and NBD-N3.     

阔口尖毛虫形成包囊期间细胞超微结构的观察

阔口尖毛虫形成囊期间,细胞质内出现条带状或管产产的内质网和由不同大小的囊泡组成的包囊壁前体。并且,前体的产生与内质网有关;细胞质内发生自噬泡消化现象,这是细胞将原有结构和能量进行贮存,利用的一种重要形式;大核向细胞质突出形成阿米巴形结构,这与大核向细胞质排出部分核物质有关。     

Effect of monoamines on the taste buds in the mouse

Summary Mouse taste buds were investigated following administration of monoamines and their precursors by fluorescence and electron microscopy. The appearance of fluorescent cells within the taste bud and the ultrastructural changes of vesicles in the gustatory cells were due to the treatment of 5-hydroxytryptophan. Small dense-cored vesicles (30–60 nm in diameter) appeared throughout the cytoplasm and accumulated especially at the presynaptic membranes of afferent synapses. Large dense-cored vesicles (80–100 nm) increased twice in number, and electron densities of their cores became more dense as compared with untreated mice. Fluorescent cells appeared in the taste bud of l-DOPA treated mice, whereas no ultrastructural changes were observed. These results suggest that the gustatory cells of the taste bud are capable of taking up and storing monoamines, which might act as neurotransmitters from the gustatory cells to the nerves.     

Colchicine-induced tubular, vesicular and cisternal organelle aggregates in absorptive cells of the small intestine of the rat. II.--Endocytosis studies

Administration of the antimicrotubular agent colchicine to adult rats (0.5 mg/100 g of body weight for 6 hr) induces formation of extended aggregates of tubular, vesicular, and cisternal organelles in the absorptive cells of the small intestine. The phosphatase reaction pattern (thiamine pyrophosphatase, acid phosphatase, acid trimetaphosphatase) suggests that the majority of them belongs to the lysosomal system (Ellinger and Pavelka, 1984). The present study extends these findings and examines the uptake and fate of intravenously injected horseradish peroxidase (HRP) at the basal and lateral cell surfaces and of intraluminally applied HRP at the apical cell surface. HRP, applied to control animals and animals pretreated with colchicine, was internalized at both apical and basolateral cell surfaces of the absorptive cells, and delivered into endosome-like vesicles, multivesiculated bodies (mvbs), dense bodies (dbs), and in several instances into Golgi cisternae. Following intraluminal application, evidence was obtained for the transport of HRP across the cell; in contrast, intravenously applied HRP was never detected at the apical cell surface. Colchicine pretreatment did not stop the uptake of HRP, which was rapidly sequestered to the clustered tubules, vesicles, and cisternae, as well as to the mvbs and dbs. After longer intervals, the portion of HRP-reactive tubules, vesicles, and cisternae within the clusters increased: 60 min after HRP-administration all of them contained HRP-activity. These results indicate that the colchicine-induced clustered organelles are recipients of endocytic materials internalized at the apical as well as at the basolateral cell surface.     

The secretory pathway of vitellogenin in the fat body of the stick insect bacillus rossius: An ultrastructural and immunocytochemical study

The fat body of the adult female stick insect Bacillus rossius was examined ultrastructurally with a view to clarifying the secretory pathway. The absence of lipid storage in the tissue allowed visualization of a polarized distribution of all organelles in the cell cytoplasm. Composite granules were distributed along the baso-apical axis of the cell according to progressive stages of maturation. At their final stage of maturation, these granules possess two distinct compartments, an electron-translucent compartment and a more electron-dense one. The origin of each of the two compartments was traced back to other organelles in the basal cytoplasm of the fat body cell. The differential origin of the two compartments contributing to the composite granules was further investigated by cytochemical analyses. Vitellogenin was detected both in the electrondense compartment of the composite granules and in the Golgi apparatus. The electron-translucent compartment of the composite granules appeared to consist mainly of urate crystals. Such enzyme activities as acid phosphatase, peroxidase and catalase were also detected in this latter compartment. The observations support the interpretation that secretion in the fat body of B. rossius entails fusion of Golgi-derived vesicles with a specialized kind of multivesicular body. While Golgiderived vesicles convey their load of newly synthesized vitellogenin to the electron-dense compartment, the multivesicular body develops the urate crystals of the electron-translucent compartment.     

Ultrastructural and Cytochemical Evidence for the Presence of Peroxisomes in the Generative Cell of Magnolia x soulangeana Pollen Grain

The generative cell (GC) development during three sequentialstages of Magnolia x soulangeana pollen grain maturation wasinvestigated by light and electron microscopy. Plastids werenot identified in this cell but mitochondria, Golgi bodies andvesicles as well as rough endoplasmic reticulum profiles werealways present. Microtubules were also present, their numberincreasing and their disposition varying during GC maturation.The most conspicuous components of the GC cytoplasm were themicrobodies. The latter were few in number in the newly formedGC, and the appearance of their matrix was different from laterdevelopmental stages. A clear microbodial proliferation occurredin the GC during an intermediate stage of pollen maturation.Then, the microbody matrix was either fibrillar to granularas in the vegetative cell microbodies or very dense and compact.The polymorphism and size range and the frequent aggregationof these organelles in one or more clusters were also noteworthy.Tilting of semithin sections as well as the analysis of serialsections suggested that a number or enlarged and irregularlyshaped microbodies co-exist with smaller and more sphericalones, the latter probably originating by budding. In the GCof the mature pollen the microbody-like organelles were in generalmore uniform both in shape and size. The cytochemical test ofDAB was positive in the microbodies of both the pollen cells,thus demonstrating their peroxisomic nature. The function ofthe microbodies in the GC is not clear. In this cell, a fewlipid droplets only exist during the first developmental stageand the microbodies were apparently unrelated to any other organelle.Possibly, these are unspecialized microbodies which are paternallytransmitted, but it is not excluded that, temporarily, theymay play some special role during GC maturation.Copyright 1994,1999 Academic Press Peroxisomes, generative cell, pollen maturation, Magnolia x soulangeana Soul.-Bod     

A Vacuolar Processing Enzyme in Maturing and Germinating Seeds: Its Distribution and Associated Changes during Development

Proprotein precursors of vacuolar components are transportedfrom endoplasmic reticulum to the dense vesicles, and then targetedto the vacuoles, where they are processed proteolytically totheir mature forms by a vacuolar processing enzyme. Immunoelectronmicroscopy of the maturing endosperm of castor bean (Ricinnscommunis) revealed that the vacuolar processing enzyme is selectivelylocalized in the dense vesicles as well as in the vacuolar matrix.This indicates that the vacuolar processing enzyme is transportedto vacuoles via dense vesicles as does IIS globulin, a majorseed protein. During seed maturation of castor bean, an increasein the activity of the vacuolar processing enzyme in the endospermpreceded increases in amounts of total protein. The enzymaticactivity reached a maximum at the late stage of seed maturationand then decreased during seed germination concomitantly withthe degradation of seed storage proteins. We examined the distributionof the enzyme in different tissues of various plants. The processingenzyme was found in cotyledons of castor bean, pumpkin and soybean,as well as in endosperm, and low-level processing activity wasalso detected in roots, hypocotyls and leaves of castor bean,pumpkin, soybean, mung bean and spinach. These results suggestthat the proprotein-processing machinery is widely distributedin vacuoles of various plant tissues. (Received July 11, 1993; Accepted August 17, 1993)     

Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface. To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [35S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP. In the presence of GTP gamma S, the constitutive vesicles became significantly more dense, suggesting that a coated intermediate was stabilized. Larger immature regulated secretory granules, enriched in sulfated secretogranin II, also escaped from the permeabilized cells in vitro. During granule maturation, their density increased and the amount of cofractionating proteoglycans diminished. The data suggest that sorting continues during secretory granule maturation.     

Ultrastructural Studies of Somatic Embryogenesis in Wheat

Electron microscopic observation of somatic embryogenesis from cultured immature wheat embryos revealed the presence of a lot of small vacuoles, a large nucleus, clear nucleolus and polynucleoli. The electron density of cytoplasm was strengthened during somatic embryogenesis. Quantity and type of organelles—plastid, ribosome and mitochondrion were increased; thickened cell wall, disappeared plasmodesmata, increased organelles andstarch accumulation in the embryogenic cells. Nucleolus vacuoles, autophagic vacuoles and secretory vesicles were present in the embryogenic cells with thickened cell walls. The multicellular proembryos, globular embryoid and pear-shaped embryoid were surrounded by an envelop, but plasmodesmata existed extensively between cells of somatic embryoid. The membranous structures appeared in the plastid which underwent transformation into chloroplast in the cells of growing point in almost mature embryoid. The relation of the above-mentioned structureal changes to somatic embryogenesis is also discussed.