TRANSFORMATION OF MONOCYTES IN TISSUE CULTURE INTO MACROPHAGES, EPITHELIOID CELLS, AND MULTINUCLEATED GIANT CELLS : An Electron Microscope Study

The sequential transformation of chicken monocytes into macrophages, epithelioid cells, and multinucleated giant cells in vitro was studied by electron microscopy after fixation and embedment in situ. The following changes occur. In the nucleus, margination of chromatin, evident in monocytes, decreases in later forms. Nucleoli become more complex and nuclear pores increase in number. In cytoplasm, a progressive increase in volume of the ectoplasm and endoplasm occurs in culture. Lysosomes increase in number and size prior to phagocytosis. During phagocytosis (most active from 1 to 3 days of culture) lysosome depletion occurs. Lysosomes are present in greatest number and show maximal structural variation in the epithelioid and young giant cells. Aging giant cells lose lysosomes. All stages possess variably large quantities of rough-surfaced endoplasmic reticulum and free ribosomes. The Golgi apparatus, small in monocytes, increases in size and complexity. Massive accumulations of lysosomes within the Golgi apparatus of macrophages and epithelioid cells suggest that lysosomes originate there. In giant cells, multiple Golgi regions occur, often ringing the nuclei. Monocytes and macrophages have few mitochondria. Mitochondria of epithelioid cells are larger, more numerous, and may have discontinuous outer membranes. Mitochondria are most numerous in giant cells where they increase with age and become polymorphous. Cytoplasmic filaments are approximately 50 to 60 A in diameter and of indeterminate length. They occur both singly and in bundles which touch cytoplasmic vesicles and mitochondria. Few filaments occur in monocytes and macrophages. A large increase in the number of filaments occurs in epithelioid cells, where filaments (90 to 100 A) surround the cytocentrum as a distinctive annular bundle often branching into the cytoplasm. The greatest concentration of filaments occurs in aged giant cells. Pseudopodia are always present. They are short and filiform in monocytes and giant cells, and broad, with abundant micropinocytotic vesicles, in macrophages and epithelioid cells. At every stage, the cell membrane contains dense cuplike structures. These may represent the membranous residue of lysosomes which have discharged to the outside, analogous to merocrine secretion. Contiguous epithelioid cells display elaborate cytoplasmic interdigitation. In places, the plasma membranes break down and epithelioid cells fuse to form giant cells.     

Histopathogenesis of Galls Induced by Meloidogyne naasi in Wheat Roots

Histopathogenesis of galls induced by Meloidogyne naasi in wheat roots was studied. Large numbers of larvae penetrated wheat root tips within 24 hr; larvae migrated both inter- and intracellularly, causing cortical hypertrophy. Giant cells were formed in the stele around the head of each nematode within 4 to 5 days. Initial pathological alterations in giant cell formation consisted of hypertrophy of protophloem and protoxylem cells, their nuclei and nucleoli. Giant ceils contained 2 to 8 agglomerated multinucleolate nuclei. Synchronous mitotic divisions were first observed 9 days after inoculation. After 21 days, giant cells became highly vacuolate. Observations 40 days after inoculation revealed a complete degeneration of cell contents in many giant cells but their thick walls remained intact. Abnormal xylem completely surrounded the degenerated or partially degenerated giant cells.     

Carapace repair in the green crab,Carcinus maenas (L.)

Formation of a circular hole 8–10 mm in diameter in the calcified layers of the carapace from crabs in stage C₄ of the molt cycle stimulates the tissue under and adjacent to the injury to deposit a unique calcified cuticular material below the intact membranous layer. Deposition was followed for 69 days using light microscopic histology, histochemistry, and scanning electron microscopy. Quantitative analyses of CaCO₃ were conducted using atomic absorption spectrophotometry and Gran titration. Spatial distribution of CaCO₃ was determined with X-radiography. A scab is formed by day two under the injury. At four days the epithelium changes from squamous to columnar and deposits a PAS-positive layer with an irregular lamellar fine structure, followed by highly organized lamellae structurally similar to normal exocuticle. Histochemically, however, these lamellae resemble normal endocuticle. CaCO₃ is evident external to the outermost lamellae by day eleven as a fused mass of aragonite granules. The lamellar region calcifies proximally from the outer surface and is amorphous CaCO₃. Repair cuticle is approximately 20%CaCO₃ by weight.     

Electron Microscopy of Monkey Kidney Cell Cultures Infected with Rubella Virus

Two rubella virus strains isolated in this laboratory were investigated in terms of their growth in LLC-MK(2) cell cultures and their effect on cell morphology. Rubella virus grew readily in LLC-MK(2) cells, but cytopathic effects of the virus were not observed in infected cultures. Such infected cultures can be subcultured indefinitely and continue to shed virus. Examination of rubella-infected cell cultures by electron microscopy showed the presence of annulate lamellae in the cytoplasm of 15% of the cells. No changes were evident in the nuclei. These membranous inclusions varied in complexity from parallel arrays of annulate lamellae to large lamellar structures of complex morphology. An occasional cell contained a crystal lattice structure in association with the lamellae. Larger inclusions, consisting of disorganized arrays of "unit" membranes, were also found. Uninfected cells were devoid of annulate lamellae, crystals, and complex membranous inclusions. No viruslike particles were observed in any part of the cells from infected cultures. The significance of the structures observed has not been determined.     

Is the nuclear envelope a ‘generator’ of membrane?

Summary Early diplotene oocytes from Necturus maculosus ranging from 0.2 to 0.5 mm in diameter were examined by electron microscopy. In the smallest oocytes of this range, the cytoplasm is largely devoid of membranes, but contains primarily ribosomes and mitochondria. In slightly larger oocytes, smooth-surfaced cytomembranes first appear in the perinuclear cytoplasm. At this time, the outer layer of the germinal vesicle nuclear envelope (GVNE) shows frequent connections with long membranous lamellae that extend for considerable, but variable distances into the juxtanuclear ooplasm. The number of smooth membranous lamellae increases tremendously as the oocytes increase in diameter. In such oocytes as well, frequent continuities are observed between the outer membrane of the GVNE and many of the cytoplasmic membranes. Eventually, as the ooplasm becomes populated with extensive numbers of membranous lamellae, instances of continuity between the membranous lamellae and nuclear envelope now become sparse and eventually non-existent. The frequent connections observed between membranous lamellae and the outer membrane of the GVNE during a circumscribed interval of diplotene strongly implicate the GVNE in the generation of extensive amounts of cytoplasmic membrane. The ooplasm of larger oocytes in the size range indicated contain numerous Golgi complexes and large quantities of annulate lamellae most of which are positioned in the peripheral or subcortical ooplasm, as well as extensive quantities of smooth membranes of the endoplasmic reticulum and lipid droplets.     

The regreening of nitrogen-deficient Chlorella fusca

Chlorella fusca, strain 211-15, cells degreened in a nitrogen-deficient mineral growth medium in the light for 4–6 weeks were regreened for up to 24 hrs in a nitrogen rich medium that leads to synchronous cell division at 24–26 hrs. Structural changes in the plastid membranes during the regreening period were observed by thin section and freeze-fracture electron microscopy. Nitrogen-deficient plastids were found to have non-appressed lamellae, prolamellar body-like membrane aggregations, and only 2 types of freeze-fracture face. At this time no photosynthetic oxygen evolution could be demonstrated. After 6 hrs regreening the plastid lamellae had fused to form bands of appressed lamellae and the four types of freeze-fracture face, described previously, were visible. At this time photosynthetic oxygen evolution could be demonstrated. After 24 hrs regreening the plastids had an appearance typical of normally grown Chlorella and had commenced to divide. Supporting evidence for these developmental stages is presented from isolated chloroplast particle fractions. An unusual type of cell wall proliferation was observed in the nitrogen-deficient Chlorella cells that resulted in the laying down of several walls, each with a trilaminar component.     

Endothelial quality of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S): two-year outcome of manual preparation in an ocular tissue bank

To assess the quantitative and qualitative parameters of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) prepared manually in the Ocular Tissue Bank Prague. All 65 successfully prepared pre-cut posterior corneal lamellae provided for grafting during a 2-year period were analyzed retrospectively. The lamellae, consisting of a central zone of endothelium-Descemet membrane surrounded by a supporting peripheral stromal rim, were prepared manually from corneoscleral buttons having an endothelial cell density higher than 2,500 cells/mm². The live endothelial cell density, the percentage of dead cells, the hexagonality and the coefficient of variation were assessed before and immediately after preparation as well as after 2 days of organ culture storage at 31 °C. Altogether, the endothelium of 57 lamellae was assessed. Immediately after preparation, the mean live endothelial cell density was 2,835 cells/mm² and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm² and the percentage of dead cells to 1.0 %. There was a significant change in the mean hexagonality and the coefficient of variation after lamellar preparation and subsequent storage. The amount of tissue wasted during the preparation was 23 %. The endothelial cell density of posterior corneal lamellae sent for DMEK-S was higher than 2,700 cells/mm² in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.     

Photoreceptor development in the pineal organ and the eye of Plecoglossus altivelis and Paralichthys olivaceus (Teleostei)

Summary The ontogenetic apperance of pineal photo-receptors was compared with that of retinal photoreceptors in the ayu Plecoglossus altivelis and the lefteye flounder Paralichthys olivaceus, which hatched 10 days and 3 days after fertilization, respectively. Despite the disparity in incubation time, the outer segments (containing membranous lamellae) of the pineal photoreceptors first appeared from 3 to 4 days after fertilization in both species. In contrast, the outer segments of the retinal photoreceptors first became visible 5 to 6 days after fertilization, although a characteristic retinal stratification and the optic tract leaving the ganglion cell layer were already found 4 days after fertilization in both species. The functional significance of these temporal disparities and/or similarities in photoreceptor development are discussed with special reference to the timing of daily rhythmic activities during the early developmental period of the teleosts.The results were presented at the Annual Meeting of the Japanese Society of Scientific Fisheries on April 2, 1990 (Tokyo)     

Life Cycle,Pathogenicity, Histopathology,and Host Range of Race 5 of the Barley Root-Knot Nematode

The optimum temperature for development of race 5 of Meloidogyne naasi was 26 C. A life cycle was completed in 34 days. Growth of sorghum was suppressed when inoculated with M. naasi. Observations of M. naasi-infected sorghum roots demonstrated that roots were penetrated just behind the root cap; giant cells were generally initiated either in the procambial region or in very young phloem. When giant cells developed in the cortex, corresponding areas of the vascular system did not have an endodermis, pericycle, or phloem fibers. Nineteen plant species were tested for suitability as hosts for race 5 of M. naasi. Reproduction occurred on 11 of 12 monocotolydenous hosts and none of 7 dicotolydenous hosts. Reproduction often occurred without gall development.     

Annulate lamellae in hemipteran preblastoderm cytoplasm and nuclei

Summary Annulate lamellae (AL) were found in preblastoderm cytoplasm and nuclei of the giant milkweed bug,Oncopeltus fasciatus. To the best of the authors' knowledge, this is the first report of such an occurrence among the insects. Cytoplasmic AL were scattered about the nucleus during early prophase and then accumulated during late prophase to form two dome-shaped arrays lying opposite each other with the nucleus interposed. Also, intranuclear annulate lamellae were found as single strands or vesicles within the nucleus during prophase.Supported in part by U.S. Atomic Energy Commission Contract AT (49-7) 3028.     

Ultrastructure of Blue-Green Algae

Two freshwater blue-green algae, Tolypothrix tenuis and Fremyella diplosiphon, and an oscillatorialike marine alga, were found to possess structures on the photosynthetic lamellae which appear to correspond to the phycobilisomes of red algae. These homologous structures are important because they contain the phycobilins which are accessory pigments involved in photosynthesis. As in the red algae, the phycobilisomes were attached on the outer side of each lamellae, i.e., the side facing away from its own membrane pair. Although our study on Anacystis nidulans has not thus far revealed the presence of phycobilisomes, some observations were made on the structure of the polyhedral bodies. After negative staining, the polyhedral bodies were seen to be composed of regularly spaced subunits arranged in a crystalline array. Elongated segmented rods, which differed from the polyhedral bodies, were found in the nuclear region of apparently healthy Tolypothrix cells.     

Ultrastructural Changes Caused by Fusarium oxysporum f. sp. lycopersici in Meloidogyne javanica Induced Giant Cells in Fusarium Resistant and Susceptible Tomato Cultivars

Tomato (Lycopersicon esculentum Mill.) seedlings, susceptible (cv. Pearson A-I Improved) and resistant (cv. Pearson Improved) to race 1 Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyd &Hans., were inoculated with Meloidogyne javanica (Trueb) Chitwood second-stage juveniles and 3 weeks later with race 1 F. oxysporum f. sp. lycopersici spores. One week after fungal inoculation, no fungus was visible in root tissue of the tomato cultivars and the giant cells were normal. Two weeks after fungal inoculation, abundant hyphae were visible in xylem tissues of Fusarium-susceptible but not of Fusarium-resistant plants. In susceptible plants, giant cell degeneration occurred, characterized by membrane and organelle disruption. In addition, where hyphae were in direct contact with the giant cell, dissolution of the giant cell wall occurred. Three weeks after fungal inoculation, fungal hyphae and spores were visible inside xylem tissues and giant cells in Fusarium-susceptible plants and in xylem tissue of the resistant plants. In susceptible and resistant plants, giant cell degeneration was apparent. Giant cell walls were completely broken down in Fusarium-susceptible tomato plants. In both cultivars infected by Fusarium, giant cell nuclei became spherical and dark inclusions occurred within the chromatin material which condensed adjacent to the fragmented nuclear membrane. No such ultrastructural changes were seen in the giant cells of control plants inoculated with nematode alone. Giant cell deterioration in both cultivars is probably caused by toxic fungal metabolites.     

The ultrastructure and cytochemistry of microbodies in dinoflagellates

Summary Microbodies, similar in morphology to those of higher plants and animals, have been observed in thirteen genera of dinoflagellates. As in other unicellular algae, they did not show any staining after incubation in DAB/H₂O₂ medium although the mitochondrial cristae and chloroplast lamellae did give a positive reaction. The microbodies of Scrippsiella sweeneyae were found to be associated with a membranous reticulate structure, which may be involved in their formation.     

Dynamics of the Nuclear Complement of Giant Cells Induced by Meloidogyne incognita

The total numbers of nuclei in giant cells induced by Meloidogyne incognita in pea, lettuce, tomato, and broad bean were determined. Mature giant cells from pea had the most nuclei per giant cell with a mean of 59 ± 23, lettuce had the fewest with 26 ± 16, and tomato and broad bean were intermediate. The rate of increase in numbers of nuclei for all plant species was greatest during the first 7 days after inoculation. No mitotic activity was observed in giant cells associated with adult nematodes. Number of nuclei per giant cell doubled each day during the period of greatest mitotic activity, but number of total chromosomes per giant cell increased 20-fold per day at the same time. The hypothesis is presented that factor(s) responsible for the polyploid, mulfinucleate condition characteristic of giant cells may be different from factor(s) responsible for aneuploid numbers of chromosome per nucleus or for nuclear aberrations such as the presence of linked nuclei.     

Endodermal cell-cell contact is required for the spatial control of Casparian band development in Arabidopsis thaliana

Background and Aims

Apoplasmic barriers in plants fulfil important roles such as the control of apoplasmic movement of substances and the protection against invasion of pathogens. The aim of this study was to describe the development of apoplasmic barriers (Casparian bands and suberin lamellae) in endodermal cells of Arabidopsis thaliana primary root and during lateral root initiation.

Methods

Modifications of the endodermal cell walls in roots of wild-type Landsberg erecta (Ler) and mutants with defective endodermal development – scarecrow-3 (scr-3) and shortroot (shr) – of A. thaliana plants were characterized by light, fluorescent, confocal laser scanning, transmission and cryo-scanning electron microscopy.

Key Results

In wild-type plant roots Casparian bands initiate at approx. 1600 µm from the root cap junction and suberin lamellae first appear on the inner primary cell walls at approx. 7000–8000 µm from the root apex in the region of developing lateral root primordia. When a single cell replaces a pair of endodermal and cortical cells in the scr-3 mutant, Casparian band-like material is deposited ectopically at the junction between this ‘cortical’ cell and adjacent pericycle cells. Shr mutant roots with an undeveloped endodermis deposit Casparian band-like material in patches in the middle lamellae of cells of the vascular cylinder. Endodermal cells in the vicinity of developing lateral root primordia develop suberin lamellae earlier, and these are thicker, compared wih the neighbouring endodermal cells. Protruding primordia are protected by an endodermal pocket covered by suberin lamellae.

Conclusions

The data suggest that endodermal cell–cell contact is required for the spatial control of Casparian band development. Additionally, the endodermal cells form a collet (collar) of short cells covered by a thick suberin layer at the base of lateral root, which may serve as a barrier constituting a ‘safety zone’ protecting the vascular cylinder against uncontrolled movement of water, solutes or various pathogens.     

Host Response to Meloidodera spp. (Heteroderidae)

Host responses to Meloidodera floridensis Chitwood et al., 1956, M. charis Hooper, 1960, and M. belli Wouts, 1973 were examined on loblolly pine, peony, and sage, respectively, with light, scanning, and transmission electron microscopy. In each case the nematodes induce a single uninucleate giant cell. The giant cell is initiated in the pericycle and expands as it matures. The mature giant cell induced by M. floridensis is surrounded by vascular parenchyma, whereas that caused by M. charts and M. belli coutacts xylem and phloem. The cell wall of giant cells induced by all three Meloidodera spp. is generally thicker than that of surrounding cells, with the thickest part adjacent to the lip region of the nematode. The thinner portion of the wall includes numerous pit fields with plasmodesmata, but wall ingrowths were not detected in a thorough examination of the entire wall. The nucleus of a giant cell induced by M. goridensis is highly irregular in shape with deep invaginations, whereas those caused by M. charis and M. belli include a cluster of apparently interconnected nuclear units. Organelles, including mitochondria, endoplasmic reticulum, and plastids of giant cells caused by Meloidodera, are typical of those reported in host responses of other Heteroderidae. The formation of a single uninucleate giant cell by Meloidodera, Cryphodera, Hylonerna, and Sarisodera, but a syncytium by Atalodera and Heterodera sensu lato, might be considered in conjunction with additional characters to determine the most parsimonious pattern of phylogeny of Heteroderidae.     

An ultrastructural study of the response of Blatella germanica (Orthoptera: Blattidae) to the nematode Abbreviata caucasica (Spirurida: Physalopteridae)

An ultrastructural study of the response of Blatella germanica (Orthoptera: Blattidae) to the nematode Abbreviata caucasica (Spirurida: Physalopteridea). International Journal for Parasitology4: 133–138. This study investigates the response of the roach, Blatella germanica L. to the invading spirurid nematode, Abbreviata caucasica v. Linstow. Soon after the first stage nematodes entered the epithelial cells of the colon wall, the surrounding host cells broke down into syncytial giant cells. Large polychromatic epithelial cell nuclei occurred throughout the giant cells and the nematodes moved freely within the cytoplasmic matrix. These giant cells were in turn surrounded by blood cells responding to the disruption. The nematodes developed to the infective third stage juveniles within the giant cells and ingested the syncytial cytoplasm. After reaching the third stage, the parasites remained in a quiescent state within the vacuolated cell which was surrounded by a double tissue layer.Evidence indicated that successful development of the parasite was dependent on the disrupted epithelial cells forming a giant syncytial cell which protected and supplied nourishment to the parasite.     

Expression and Regulation of the Arabidopsis thaliana Cel1 Endo 1,4 �� Glucanase Gene During Compatible Plant-Nematode Interactions

The root-knot nematode Meloidogyne incognita is an obligate endoparasite of plant roots and stimulates elaborate modifications of selected root vascular cells to form giant cells for feeding. An Arabidopsis thaliana endoglucanase (Atcel1) promoter is activated in giant cells that were formed in Atcel1::UidA transgenic tobacco and Arabidopsis plants. Activity of the full-length Atcel1 promoter was detected in root and shoot elongation zones and in the lateral root primordia. Different 5’ and internal deletions of regions of the 1,673 bp Atcel1 promoter were each fused to the UidA reporter gene and transformed in tobacco, and roots of the transformants were inoculated with M. incognita to assay for GUS expression in giant cells and noninfected plant tissues. Comparison of the Atcel1 promoter deletion constructs showed that the region between −1,673 and −1,171 (fragment 1) was essential for Atcel1 promoter activity in giant cells and roots. Fragment 1 alone, however, was not sufficient for Atcel1 expression in giant cells or roots, suggesting that cis-acting elements in fragment 1 may function in consort with other elements within the Atcel1 promoter. Root-knot nematodes and giant cells developed normally within roots of Arabidopsis that expressed a functional antisense construct to Atcel1, suggesting that a functional redundancy in endoglucanase activity may represent another level of regulatory control of cell wall-modifying activity within nematode feeding cells.     

Histological Responses of Four Leguminous Crops Infected with Meloidogyne incognita

Histological responses to Meloidogyne incognita infection in Rhizobium nodules of clover, horsebean, lupine, and pea were investigated. The formation of giant cells in vascular bundles of nodules and roots, and the basal connection of the nodule, were usually associated with abnormal xylem and/or deformed xylem strands. However, giant cells did not disturb or prevent the development of nodular tissues. Areas in which galls formed, wall thickness of giant cells, and number of giant cells around the nematode head varied with plant species. Ranking by gall size and giant-cell wall thickness was horsebean > lupine and pea > clover. The multinucleate condition in giant cells resulted from repeated mitoses without subsequent cytokinesis. The resulting nuclei agglomerated in irregularly shaped masses in some giant cells.