Anatomy and ultrastructure of epidermal cells in the haustorium of a parasitic flowering plant,Cuscuta japonica, during attachment to the host

Changes of epidermal cells in the haustorium of the parasiticCuscuta japonica during its attachment to the host plantimpatiens balsamina were studied with light and electron microscopy. In the transverse sections of dodder stems not in contact with the host, epidermal cells had rounded outlines. However, when haustorial initials developed in the cortex of the parasite stem at the contact site, the epidermal cells had more dense cytoplasm and conspicuous nuclei than before, and their outline was flat in the longitudinal section. As meristem cells developed from those initials, the epidermal cells became more elongated. When the haustorium was fully matured, the apical tips of the elongated epidermal cells at the contact site branched like toes, producing numerous projections via cell wall invaginations. This event caused spaces to form between the projections; coincidently, the surface area of the apical ends of the epidermal cells increased. The dense cytoplasm at those projections contained prominent nuclei and abundant other organelles, suggesting a active metabolism. Osmiophilic particles, releasing into the cell walls from the cytoplasm, were though to be associated with the loosening and elongating of the epidermal cell walls. Dense and homogeneous materials were secreted within the spaces between the projections. These materials could play an important role in cementing the haustorium onto the surface of the host organ.     

Fine structure of fusion products from soybean cell culture and pea leaf protoplasts

Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.Abbreviations PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy Supported by National Research Council of Canada, Grant A6304     

Leaf‐shape remodeling: programmed cell death in fistular leaves of Allium fistulosum

Some species of Allium in Liliaceae have fistular leaves. The fistular lamina of Allium fistulosum undergoes a process from solid to hollow during development. The aims were to reveal the process of fistular leaf formation involved in programmed cell death (PCD) and to compare the cytological events in the execution of cell death to those in the unusual leaf perforations or plant aerenchyma formation. In this study, light and transmission electron microscopy were used to characterize the development of fistular leaves and cytological events. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) assays and gel electrophoresis were used to determine nuclear DNA cleavage during the PCD. The cavity arises in the leaf blade by degradation of specialized cells, the designated pre‐cavity cells, in the center of the leaves. Nuclei of cells within the pre‐cavity site become TUNEL‐positive, indicating that DNA cleavage is an early event. Gel electrophoresis revealed that DNA internucleosomal cleavage occurred resulting in a characteristic DNA ladder. Ultrastructural analysis of cells at the different stages showed disrupted vacuoles, misshapen nuclei with condensed chromatin, degraded cytoplasm and organelles and emergence of secondary vacuoles. The cell walls degraded last, and residue of degraded cell walls aggregated together. These results revealed that PCD plays a critical role in the development of A. fistulosum fistular leaves. The continuous cavity in A. fistulosum leaves resemble the aerenchyma in the pith of some gramineous plants to improve gas exchange.     

Ultrastructure and function of nurse cells in phthirapterans. Possible function of ramified nurse cell nuclei in the cytoplasm transfer

The structure of nurse cells as well as the distribution of cytoskeletal elements (actin filaments, microtubules) in three representatives of phthirapterans: the pig louse, Haematopinus suis (Anoplura) and bird lice, Eomenacanthus stramineus, Columbicola columbae (Mallophaga) were investigated. All three species have polytrophic-meroistic ovaries which means that each oocyte remains connected with a group of nurse cells via specialized cytoplasmic canals-intercellular bridges (ring canals). Throughout vitellogenesis, various macromolecules as well as organelles (mitochondria, endoplasmic reticulum vesicles, ribosomes) are transferred from the nurse cells to the oocyte. During this flow, the nurse cell nuclei do not enter the oocyte and are retained in the cell centers. In holometabolous insects (e.g. Drosophila, hymenopterans), the central position of nurse cell nuclei is maintained by cytoskeletal elements (actin filaments or microtubules). In the investigated species, the nurse cells are equipped with large, highly extended (irregularly lobed) nuclei. The inner nuclear membrane is lined with a relatively thick layer of nuclear lamina. Ultrastructural analysis and staining with rhodamine-labeled phalloidin revealed that the nurse cell cytoskeleton is poorly developed and represented only by: (1) single microtubules in the perinuclear cytoplasm; and (2) the F-actin layer in the cortical cytoplasm. In the light of this, we postulate that in phthirapterans the position of nurse cell nuclei during the cytoplasm transfer is maintained not by the cytoskeletal elements, but by a largely extended shape of the nuclei (i.e. their elongated extensions).     

Ultrastructure of tapetal cell ontogeny inBeta

Summary Tapetal cell development and degeneration in anthers ofBeta vulgaris L. were studied with the electron microscope. Tapetal cells become differentiated from sporogenous cells early in anther ontogeny. The tapetal nuclei divide mitotically; binucleate tapetal cells contain relatively little endoplasmic reticulum and otherwise resemble meristematic cells of higher plants. There follows an increase in endoplasmic reticulum and by the time the sporogenous tissue has entered meiotic prophase, the tapetal cells have differentiated the usual characteristics of secretory cells. Degenerative changes begin to appear in tapetal cells after meiosis of the sporogenous tissue. Such changes include loss of inner tangential and anticlinal walls, degeneration of tapetal nuclear envelopes, disruption of the plasmalemma, and changes in the cytoplasmic organelles. Coated tubules are associated with tapetal nucleoli during degenerative stages and the tubules persist after tapetal nuclei have degenerated. Tapetal cell cytoplasm disappears completely by the stage of microspore mitosis.     

Immunocytochemical localisation of auxin-binding proteins in coleoptiles and embryos ofZea mays L.

Summary The auxin-binding protein ABP-1 was localised immunocytochemically in coleoptiles and immature embryos ofZea mays. Two primary polyclonal antibodies raised against ABP-1 and secondary antibodies were either labelled with FITC or 10 nm gold particles for light microscopy, and with 10 nm gold particles for transmission electron microscopy. Light microscopy revealed that ABP-1 was localised in the epidermal cells of etiolated maize coleoptiles, in subepidermal parenchymatic mesophyll cells of the coleoptile and in the companion cells of the vascular bundles. Most labelling was found in the cytoplasm, less in nuclei and vacuoles and cell walls appeared negative. The region of the plasma membrane exhibited prominent labelling. Embryos showed low labelling throughout their tissues just after excision, but after culture for 7 days intensive labelling was found in the epidermis of the scutellum. Quantitative electron microscopy confirmed that ABP-1 was present in the cytoplasm of epidermal, mesophyll, and companion cells of coleoptiles. Gold particles were neither found in cell walls nor in the cuticle. Areas with ER and dictyosomes within epidermal and mesophyll cells of coleoptiles had a denser labelling with gold particles than elsewhere. Labelling at the plasma membrane, being the site where the auxin binds to the ABP, was observed at low levels in all cells examined, which is due to the method applied. Epidermal cells of embryos cultured for 5 days exhibited high levels of gold particles in ER and nuclei, and lower levels in the cytoplasm. The distribution is only partly in accordance with the model in which ABP is thought to cycle through the plant cell from the ER via the Golgi system towards the plasma membrane.Abbreviations ABP-1 auxin-binding protein 1 - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - LM light microscopy - LR Write London resin white - PBS phosphate-buffered saline - PEG polyethylene glycol - TEM transmission electron microscopy     

Light microscopic detection of PAS-positive substances with thiosemicarbazide in freeze-substituted ovaries of Paspalum longifolium before pollination

Summary Localization of polysaccharides in the freeze-substituted, Eponembedded ovaries of Paspalum longifolium prior to pollination was carried out by periodic acid-Schiff's (PAS), periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-AgPr) and periodic acid-thiosemicarbazide-osmium (PA-TSC-OsO₄) reactions. The specificities of these three reactions were also studied. These three reactions are all effective for light microscopic demonstration of polysaccharides in the filiform apparatus, starch grains in the cells and PAS substance in the micropylar region. Nonspecific staining of the nucleoli of the egg and polar nuclei was observed in the PAS reaction. The PA-TSC-AgPr reaction is very specific for polysaccharides but its overall reaction takes a much longer period of time than the PAS reaction. The PA-TSC-OsO₄ reaction colors the cytoplasm and nuclei of most cells and therefore stains of the cell walls, especially those of the egg cell and synergids, do not stand out clearly. The synergid cytoplasm contains some amorphous polysaccharides and thus it colors even in PAS and PA-TSC-AgPr preparations. In the mature embryo sac, the egg and central cell as well as antipodals are vacuolated but the two synergids have no visible vacuoles under light microscope. Each synergid has a prominent filiform apparatus at the micropylar end, which stains intensely in all three preparations. The walls of the central cell and antipodals adjacent to the nucellar cells have many inward papillae which are also intensely stained in all three preparations. Starch grains are abundant in the ovary wall and usually absent in the nucellus and integuments. They are present in the egg, central cell and antipodals, but not in the two synergids.     

Establishment and characterization of a new human renal cell carcinoma cell line (KRC/Y)

Summary A new renal cell carcinoma (RCC) cell line (KRC/Y) has been established from a surgical specimen of a 41-yr-old Japanese female patient with RCC composed of both clear cells and granular cells. This cell line has been maintained for more than 15 mo. through 45 passages with a stable growth, KRC/Y cells have clear or eosinophilic polygonal cytoplasm and round to oval nuclei with one or two nucleoli, and proliferate in a pavementlike cell arrangement with a lack of contanct inhibition. By electron microscopy, these cells contain abundant fat droplets and glycogen granules or well-developed organells or both, which were also observed in the original tumor. The doubling time of these cells at the 15th passage was 73 h. The chromosome number was from 37 to 45 with a hypodiploid modal number of 42. Tumorigenicity was identified by tumor formation after subcutaneous injections of KRC/Y cells in nude mice, which showed close resemblance to the original tumor by light and electron microscope observations. This study was supported in part by Sarah Cousin Fund, Boston, MA.     

Programmed cell death during pigment gland formation in Gossypium hirsutum leaves

Ultrastructural studies have shown that the formation of pigment glands in Gossypium hirsutum L. leaves is a lysigenous process, originating from a cluster of cells in the ground meristem. Various techniques were used here to investigate whether programmed cell death (PCD) plays a critical role in this developmental process. Nuclei of internal cells in the pigment gland‐forming tissue were TUNEL‐positive and DAPI‐negative, suggesting that DNA cleavage is an early event and complete DNA degradation is a late event. Smeared bands and a lack of laddering after gel electrophoresis indicate that DNA cleavage is random. Ultrastructurally, secretory cells in the pigment glands become distorted, nuclei are densely stained, and chromosomes become condensed until completely degraded at late stages. Vacuoles with electron‐dense bodies and membrane‐bound autophagosomes are seen in both secretory and sheath cells, suggesting that autophagy plays a key role in PCD during cytoplasm degradation. Buckling of cell walls, seen at early stages, later leads to a complete breakdown of the walls. Together, these results suggest that PCD plays a critical role in the lysigenous development of pigment glands in G. hirsutum leaves.     

Anatomical and ultrastructural study of the secretory cavity development ofCitrus sinensis and Citrus limon: evaluation of schizolysigenous ontogeny

The ontogeny and ultrastructure of secretory oil glands in the fruits of Citrus sinensis and Citrus limon were studied with light and electron microscopy (TEM). Oil glands were initiated by cell division in globular/oval clusters of young meristematic cells under or including also the epidermis. Successively, the central oil cells increased greatly in size by growth and coalescence of one or more vacuoles. Starting from these cells the oil cavity formation and enlargment occurred by schizogenous and lysigenous precesses which overlap one another. The separation of the cell walls, in both Citrus species, was accompanied by strong structural disorganization of the cytoplasm, loss of nuclei, plasmolysis, and followed by autolysis processes. Prior these changes a great quantity of oil droplets filled the cells. In Citrus sinensis this material was formed in the plastids; on the contrary, in Citrus limon oil droplets appeared only in the cytoplasm, and no connection with the plastids was observed. The processes of schizogeny and lysigeny occurred in a centrifugal direction. The peripheral oil chamber cells were so pushed outward and flattened with the progressive expansion of the gland cavity. Also these cells became vacuolated, showed schizogenous processes together with cytoplasm and nuclei breakdown. These cell modifications were preceded by an abundant essential oil synthesis. Electron dense material appeared in the cavity near the boundary cells. Among the cell layers surrounding the cavity, the outer peripheral cells showed very thick walls. These cells, probably, had a protective function, while the inner cells continued, at least in part, to produce and secrete oil compounds. In the two Citrus species the pattern of oil gland development was practically the same, except the different plastid behaviour.     

An electron microscopic study of the mature megagametophyte in Zea mays

With light microscopy maize megagametophytes stained with Alcian blue-periodic acid-Schiff (AB-PAS) reveal acid or neutral polysaccharides in various cell walls. Comparative fine structural studies were made of permanganate- or OsO₄-fixed material. Organelle distribution is random in the vacuolate and multinucleate antipodal cells; organelles are abundant; starch is scarce. Antipodal cell walls have large openings forming several syncytia. Some walls are papillate. In the central cell (primary endosperm cell) a thin peripheral layer of cytoplasm surrounds the large vacuole; organelle number is moderate; starch is abundant. The central cell wall is also papillate adjacent to the antipodals and around the egg apparatus. In the synergids organelle distribution is non-random; nuclei and numerous organelles occupy the micropylar cytoplasm of each synergid; vacuoles dominate the chalazal cytoplasm of these cells. The filiform apparatus stains with AB-PAS and is composed of both lightly and darkly stained amorphous material. In the egg, organelle distribution is perinuclear with vacuoles proximal to the micropyle; mitochondria are large, abundant and polymorphic; starch is abundant. Nucleolar diameter is five times greater in the central cell and egg than in the antipodal cells and ten times greater than in the synergids. Plasmodesmata occur in all cell walls within the gametophyte, but none appear in the gametophyte wall itself. It is suggested that the antipodals and synergids might be secretory, the latter probably being involved in pollen tube attraction, and that stored metabolites in the central cell and egg cytoplasm support rapid increase in metabolism following fertilization.     

The Structure and Origin of the Plasmodium of Rhopalura ophiocomae(Phylum Orthonectida)

Abstract Adult orthonectids develop from germinal cells within a cytoplasmic matrix called a plasmodium. This is generally assumed to be formed by the parasite. In the case of Rhopalura ophiocomae, which lives in the brittle star Amphipholis squamata, the plasmodia occupying the perivisceral coelom are closely associated with the walls of the genital bursae or the gut, and they are covered by peritoneum. They have been reported to contain scattered small nuclei distinct from those within germinal cells, embryos, and adults, but the results of the present study indicate that such nuclei probably do not exist. Furthermore, electron micrographs show that some plasmodia are in continuity with the cytoplasm of contractile cells that lie beneath the peritoneum of a genital bursa or the gut of the host. The matrix of a plasmodium of R. ophiocomaeappears, therefore, to consist of cytoplasm of a contractile cell. It is proposed that after a contractile cell has been entered by an infective cell of the parasite, it hypertrophies, bulging progressively farther into the perivisceral coelom and lifting up the peritoneum, which remains in intimate contact with it.     

Light-dependent PER-like proteins in the cephalic ganglia of an apterygote and a pterygote insect species

Antibodies targeted to a highly conserved tetradecapeptide region of the pivotal biological clock protein PER detect in the firebrat Thermobia domestica a 115-kDa protein and in the cockroach Periplaneta americana a 110-kDa protein that are present in the cytoplasm of a small set of brain cells. A similar cytoplasmic reaction occurs with antisera to the whole PER protein of Drosophila melanogaster, but these antisera also react with numerous cell nuclei. On western blots, they detect an 80-kDa antigen in T. domestica and 70- and 80-kDa antigens in P. americana. No indication of antigen translocation between cell nuclei and cytoplasm was found. Nuclear staining is maintained at a high constant level in T. domestica held at a 12:12 h light:dark photoperiod (LD) or in continuous light, but disappears rapidly in response to extended darkness. In P. americana under LD conditions, the number of immunoreactive nuclei and their staining intensity fluctuate in parallel, with maximal staining late in the day. The circadian changes are maintained in continuous light but all staining vanishes in continuous darkness. A 6-h light pulse in early night of an LD cycle induces maximal staining after about 10 h, suggesting that the effect of light on nuclear PER-like expression is indirect. The behaviour of nuclear antigens is opposite to that of the cytoplasmic PER-like proteins that persist in constant darkness and disappear in constant light. Under LD conditions, the cytoplasmic PER-like antigen cycles in T. domestica but remains at a steady level in P. americana. The sensitivity to photoregime suggests that both the nuclear and the cytoplasmic PER-like antigens are components of the biological clock.R. Závodská and H. Sehadová contributed equally to this work     

A direct light effect on maintaining photosynthetic activity of Nitella chloroplasts

The chloroplasts of internodal cells of Nitella are fixed to a stationary layer of cytoplasm whereas the nuclei and most of the cytoplasm stream along the longitudinal axis. Isolated internodal cells were maintained for several days with half the cell kept in the dark, the other half kept under continuous light. Photosynthetic activity of the cells was checked by placing the cell evenly illuminated in a ¹⁴CO₂ atmosphere. Chloroplasts of the previously dark half of the cell were found to fix only half as much CO₂ as the chloroplasts which were continuously illuminated. These results are discussed in relation to the possible direct effect of light on biosynthetic reactions of mature chloroplasts.     

Programmed cell death in the larval salivary glands of <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera,Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.     

Side branch formation and orientation in the caulonema of the moss,Funaria hygrometrica: Normal development and fine structure

Summary The regular branching of theFunaria caulonema filaments is partly related to rhythms in nuclear and cell division. The formation and development of the branches were studied by light and electron microscopy with particular attention directed to the distribution of microtubules and the polar organization of the cytoplasm. The new side branch breaks through the wall of the mother cell. The site of branch development is determined by the position of the nucleus of the mother cell. In protonemata which grow in vertically placed Petri dishes gravity influences the position of nuclei and side branches, and also the direction of oblique cross walls in the caulonema filaments to a certain extent.     

Variation in buoyant density of whole cells and isolated cell walls of Streptococcus faecium (ATCC 9790).

The buoyant density of whole cells of Streptococcus faecium varies with growth rate and during the cell cycle. Two possible explanations for this were explored: (i) the density of cell walls may vary, and (ii) the proportions of wall and cytoplasm may vary. We tested the first possibility by isolating walls from chilled, unfixed populations of S. faecium cells and fractionating them on Percoll density gradients. Mean cell wall density averaged 4% less than whole-cell density and did not vary significantly with growth rate. In addition, walls isolated from heavy and light fractions of a population of cells did not differ significantly in density. Thus, variation in the density of isolated cell walls could not account for the observed variation in whole-cell density within or between populations. Using previously published measurements of the physical dimensions of S. faecium cells, we also found that the relative proportions of wall and cytoplasm (see the second possibility above) could not account for the observed changes in whole-cell buoyant density.     

嫌色性肾细胞癌与嗜酸细胞瘤的临床病理分析与比较

目的探讨嫌色性肾细胞癌(chromophobe renal cell carcinoma,CRCC)和嗜酸细胞瘤(oncocytoma)的临床病理学特征,提高对二者的认识和诊疗水平。方法对手术切除的5例CRCC和2例Oncocytoma进行肉眼和光镜观察、免疫组织化学染色,分析其临床病理学差异。结果 CRCC以实体结构为主、胞质半透明或嗜酸性颗粒状、核皱缩且核周有空晕;Oncocytoma以巢状结构为主,胞质嗜酸颗粒状、核圆形、核仁小。免疫组化染色显示两者均呈E-cadherin、EMA、CK20、vimentin阳性;CRCC瘤细胞CD10和CK7强阳性而S-100蛋白阴性,5名患者随访8~32个月,其中1例手术12个月后由于转移死亡;Oncocytoma瘤细胞S-100蛋白强阳性、CD10和CK7阴性,2名患者随访41个月,均未出现复发和转移。结论 CRCC和oncocytoma二者形态学各有特征,免疫表型相似,二者鉴别诊断主要在于生长方式和细胞学特征。综上所述:CRCC为惰性肿瘤,Oncocytoma为良性肿瘤,预后较好,手术切除后极少发生复发和转移。     

Fine structure of the phylogenetically important marine algaRhodogorgon carriebowensis (Rhodophyta,Batrachospermales?)

Summary The fine structure of the recently described red algaRhodogorgon carriebowensis J. Norris et Bucher was studied by light microscopy and scanning and transmission electron microscopy to aid in the ordinal placement of this unusual alga. Most significant in this context were findings that pit plugs had two-layered plug caps, the outer layer of which formed a large dome and was composed of glycoprotein. Cap membranes appeared to be absent. Medullary cells were remarkable in having extremely thick, layered cell walls, whose inward deposition left little room for cytoplasm. Medullary filaments branched little except near the base of the cortex. The assimilatory filaments that made up most of the cortex contained almost all the pigmentation and starch reserves of the thallus. These filaments terminated in either slender apical cells with smooth cell walls or bulbous apical cells bearing spinulose projections. Two types of elongated cells were found in the cortex, those with calcified cell walls and those surrounded by multiple, spreading layers of wall material. Neither cell type was pigmented. The latter type sometimes supported normal assimilatory cells.The hypothesis is proposed thatRhodogorgon is a descendant of the marine progenitors of the Batrachospermales and thus is a member of this order.Abbreviations DIC differential interference contrast - PAS periodic acid Schiff - PTA-CA phosphotungstic acid-chromic acid