Kinetic and Morphological Observations on Saccharomyces cerevisiae During Spheroplast Formation

A strain of Saccharomyces cerevisiae which produced elongated cells under our growth conditions was investigated. By digestion of the cell walls with snail enzyme, the cells became spheroplasts after a transient state which we termed "prospheroplast." The prospheroplast could be lysed like the spheroplast, but it retained the shape of the original yeast cell if osmotically protected. Prospheroplasts and spheroplasts were prepared, and thin sections of samples taken throughout the process of wall removal were studied in the electron microscope, at regular intervals up to the time of complete conversion to spheroplasts. In addition, cell wall remnants recovered from spheroplast preparations were shadow cast for electron microscopy. This material revealed structures resembling bud scars with attached membranous matter. The kinetic studies showed that after a certain period of time all cells were transformed into prospheroplasts, whereas spheroplast formation started later, depending on the enzyme concentration. In sections, the prospheroplasts appeared to be formed by detachment of the cell walls. Both the prospheroplasts and the spheroplasts showed asymmetric cytoplasmic membranes in which the outer leaflets appeared coated with a dense fibrillar layer. The experiments suggest that, after enzyme digestion, the cytoplasmic membrane retains a coating which is rigid in the prospheroplast but which loses rigidity when the cell is transformed into a spheroplast.     

Effect of alkali on the structure of cell envelopes of Chlamydia psittaci elementary bodies.

Suspensions of isolated cell envelopes of infectious elementary bodies (EB) of Chlamydia psittaci at alkaline pH showed a rapid, extensive decrease in absorbance, accompanied by the release of a cell envelope component in a sedimentable form. This phenomenon was observed both at 0 C and with envelopes which had been previously heated to 100 C. Monovalent and divalent cations effectively inhibited the turbidity loss, whereas ethylenediaminetetraacetate (EDTA) caused an accelerated decrease in turbidity. The turbidity loss observed after incubation of the envelopes at alkaline pH could be reversed to the level of the initial value by dialysis against distilled water containing Mg2+. Thin-section electron photomicrographs of purified EB exposed to alkaline buffer with EDTA revealed the loss of the internal contents of cells, but these cells still maintained their round shapes. The cell surface of treated EB appeared pitted in negatively stained preparations, whereas intact EB had a smooth surface. Electron microscopic studies on negatively stained preparations of the clear supernatant obtained after the treatment of the envelope with alkaline buffer containing EDTA demonstrated the presence of spherical particles, approximately 6 to 7 nm in diameter, and rodlike particles, which appeared to be made up of two or more spherical particles.     

THE CELL ENVELOPES OF TWO EXTREMELY HALOPHILIC BACTERIA

The cell envelope of Halobacterium halobium was seen in thin sections of permanganate-fixed cells to consist of one membrane. This membrane appeared mostly as a unit membrane but in a few preparations it resembled a 5-layered compound membrane. The cell envelope of Halobacterium salinarium at high resolution was always seen as a 5-layered structure different in appearance from the apparent compound membrane of H. halobium. The "envelopes" which were isolated in 12.5 per cent NaCl from each organism were indistinguishable from each other in the electron microscope and comprised, in each case, a single unit membrane with an over-all thickness of about 110 A. Some chemical analyses were made of isolated membranes after freeing them from salt by precipitating and washing with trichloroacetic acid. Such precipitated membranes consisted predominantly of protein, with little carbohydrate and no peptido-aminopolysaccharide (mucopeptide). Sectioned whole cells of H. halobium contained intracellular electron-opaque structures of unknown function.     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

Isolation, Characterization, and Ultrastructure of the Peptidoglycan Layer of a Marine Pseudomonad

The peptidoglycan layer of a marine pseudomonad was observed by electron microscopy in thin sections of plasmolyzed intact cells and mureinoplasts but not in untreated intact cells. Only fragments of this layer could be isolated by sodium lauryl sulfate (SLS) treatment of mureinoplast envelopes. Sacculus-like peptidoglycan structures were obtained from growing cells by immediate heat inactivation of cellular autolytic enzymes and subsequent SLS, trypsin, and nuclease treatments. Recently, similar peptidoglycan sacculus-like structures have been obtained by adding SLS to the growing culture and treating the isolated particulate material with nucleases. Thin-sectioned and negatively stained preparations of whole cell peptidoglycan showed compressed profiles of cell-shaped sacculi. Peptidoglycan prepared by SLS treatment of mureinoplast envelopes had a similar composition to that prepared from whole cells. The major amino sugars and amino acids in the peptidoglycan component were glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in the molar ratios 1.18:1.24:1.77:1.00:0.79. Forty-five per cent of the epsilon-amino groups of diaminopimelic acid were cross-linked. The peptidoglycan was estimated to account for about 1% of the cell dry weight.     

A histone 1-like antigen is a component of the nuclear envelope

Rabbit antibodies have been raised against rat liver nuclear envelopes. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titer antiserum specific for the nuclear envelope preparation. Immunocytochemical studies showed that the antiserum stained the nuclear envelopes, but not intra-nuclear components of HEp-2 (human malignant epithelial) cells. When electrophoretically separated peptides were tested by immunoblotting techniques, the rabbit antiserum specifically stained proteins with molecular masses of 26 and 28 kD. These peptides had similar mobilities to purified histone 1 (H1). Indeed purified calf thymus H1 recognized the antiserum. The antigens are not loosely bound to the nuclear envelope, as they could not be extracted with low salt. Therefore, we have established that the 26 and 28 kD nuclear envelope peptides are not contaminants of the nuclear envelope preparation and that they express determinants that are immunologically cross-reactive with purified H1, but not with intra-nuclear H1.     

Envelope formation and structure in the Euglenoid genus Trachelomonas

Ultrastructural details of envelope formation are described comparatively for several species of the genus Trachelomonas. The first-formed envelope component in T. oblonga var. punctata Prings. is an external “skin”. Next, a fibrillar layer is produced between the skin and the cell surface. Ferric hydroxide and manganese compounds are precipitated on this inner layer to form a thick envelope, species-specific in size, shape, ornamentation and collar characteristics. The original skin is then lost.

A similar sequence of events occurs during envelope formation in other species, but differences exist in the details of the process and in the texture and complexity of construction of the mature envelopes. T. volvocina Ehrenb. has a fibrillar inner layer and granular precipitation, but produces a complex three-layered envelope with a honeycombed central layer. This is compared with the superficially similar three-layered envelope of an unnamed species from a wild collection, and subtle differences in ultrastructural patterning are discussed. T. pertyi Prings. forms a two-layered spiny envelope, less highly organised than that of T. volvocina. The spiny and porous envelopes of T. hispida (Perty) Stein em. Defl., T. echinata Singh and T. lefevrei Defl. are quite different in texture, being composed of flaky material woven into a spongy matrix. The presence of large amounts of iron in the mature envelopes of all the species under study was confirmed by microanalysis in the electron microscope using X-ray spectroscopy.

Details of envelope ultrastructure are discussed in relation to taxonomy, cell metabolism, the problem of why Trachelomonas cells form envelopes when those of other euglenoid genera do not, and the problem of cell control of species-specificity of envelope characters. Electron microscopy has added considerably to our knowledge of the structural details of trachelomonad envelope formation, but it has been less successful in helping to elucidate some of the enigmas associated with this phenomenon.     

Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components

Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings," "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 +/- 11 MD; intact NPC without plug, 112 +/- 11 MD; heavy ring, 32 +/- 5 MD; light ring, 21 +/- 4 MD; plug-spoke complex, 66 +/- 8 MD; and spoke complex, 52 +/- 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.     

Ultrastructure of Treponema microdentium and Borrelia vincentii

Bladen, Howard A. (National Institute of Dental Health, Bethesda, Md.), and Edward G. Hampp. Ultrastructure of Treponema microdentium and Borrelia vincentii. J. Bacteriol. 87:1180-1191.-A small oral Treponema (FM) and Borrelia vincentii (N9) were harvested after 3 to 7 days of incubation and either embedded in Vestopal W or negatively stained with phosphotungstate. The protoplasmic cylinders of both strains were identical except for size, and had a triple-structured cell wall as well as intracellular concentric laminations. Protoplasmic cylinders of both strains were enclosed in a cell envelope which appeared amorphous in negatively stained preparations, but which had a triple-structured wall when viewed in thin sections. The cell envelope of strain FM also acted as an envelope for the terminal filament; no filament envelope was evident in strain N9. Large structures which contained variable numbers of organisms and which were representative of spirochetal granules were observed. Protoplasmic cylinders contained within such granules frequently were devoid of cell envelopes. The axial filament consisted of several individual fibers which usually terminated in small end knobs. Occasionally, a fiber of the axial filament became a fiber of the terminal filament. Fibers of the terminal filament originated in end knobs similar to, but separate from, those to which the axial filament was attached. A periodicity of 60 A was occasionally observed in the terminal filament envelope of strain FM. A microperiodicity of approximately 20 A was also observed. The fibers of the terminal filament of strain N9 were composed of a large number of fibrils approximately 15 A wide. The periodicity and fibrillar structure of the terminal filament is discussed with reference to proposed models of bacterial flagella suggested by X-ray diffraction data.     

Basal structure and attachment of flagella in cells of Proteus vulgaris

Abram, Dinah (Purdue University, Lafayette, Ind.), Henry Koffler, and A. E. Vatter. Basal structure and attachment of flagella in cells of Proteus vulgaris. J. Bacteriol. 90:1337-1354. 1965.-The attachment of flagella to cells of Proteus vulgaris was studied electron microscopically with negatively stained and shadow-cast preparations of ghosts from standard cultures and from special cultures that produced "long forms." The flagellum, the basal portion of which is hooked, arises within the cell from a nearly spherical structure, 110 to 140 A in diameter. This structure appears to be associated with the cytoplasmic membrane; it may be a part of the membrane or a separate entity that lies just beneath the membrane. Flagella associated with cell walls free from cytoplasmic membrane frequently have larger bodies, 200 to 700 A in diameter, associated with their base. These structures probably consist at least partly of fragments of the cytoplasmic membrane, a portion of which folds around a smaller structure. Flagella in various stages of development were observed in long forms of P. vulgaris cells grown at low temperature. The basal structure of these flagella was similar to that of the long or "mature" flagella. Strands connecting the basal structures were observed in ghosts of long forms; these strands appear to be derived from the cytoplasmic membrane. Flagella were found to be attached to fragments of cell wall and to cytoplasmic membrane in a similar manner as they are attached to ghosts. In isolates of flagella that have been separated from the cells mechanically, the organelles often terminate in hooks which almost always appear naked, but have a different fine structure than the flagellum proper.     

鸭肠炎病毒CHv强毒株超微结构研究

将鸭成纤维细胞培养的鸭肠炎病毒(DEV),经超声处理、高速冷冻差速离心后,采用酒石酸钾—甘油非线性密度梯度超速离心,收集病毒蛋白带,3%磷钨酸负染后观察病毒粒子形态。结果表明:病毒粒子主要集中在40%~50%酒石酸钾—甘油缓冲液交界层。电镜下,病毒粒子纯净,具有疱疹病毒典型形态结构,剖面六角,外观轮廓清楚。成熟病毒粒子直径约150~266nm,病毒囊膜、核衣壳和核心清晰可见;囊膜外层较内层着色略深,且可见尚未形成完整囊膜的柄状拖尾结构。多数病毒粒子以单核衣壳为主,一定数量的病毒具有双核衣壳,偶见三核衣壳,核衣壳直径为100~150nm,呈现致密圆形、半圆形或马蹄形等类型。在核衣壳外和囊膜之间可见明显的亮晕。核心DNA电子染色较深,集中分布,直径40~65nm。本文获得的清晰DEV负染超微结构照片,为该病毒结构生物学的研究提供了重要依据。     

A rapid high-yield isolation method for nuclear envelope ghosts

A method is presented for the isolation of nuclear envelopes from isolated Tetrahymena macronuclei. In principle, nuclei are treated with DNase and RNase at low Ca²⁺/Mg²⁺ concentration followed by an extraction with 1 NaCl. The major advantages of this method are: (1) Unfragmented nuclear envelopes are obtained in the form of ghosts consisting of two juxtaposing nuclear membranes interrupted by pores as revealed by thin-section and freeze-etch electron microscopy. (2) The ghosts are obtained in high yield (60%) within a short period (1 h). (3) The nuclear envelopes largely retain their lipid composition. An average ghost contains about 96% of total phospholipids of an average nucleus. Nuclei and ghosts reveal an almost identical pattern of phospholipids and fatty acids as shown by thin-layer and gas-chromatography. (4) The lipids in the ghosts largely remain arranged in bilayers as probed by electron spin resonance using 5-doxylstearic acid as a spin label.     

SOME DYNAMIC ASPECTS OF THE NUCLEAR ENVELOPE

Nuclei of frog oocytes were isolated, fixed in OsO₄ or KMnO₄, and washed. Nuclear envelopes were then dissected off, placed on grids, and air-dried for electron microscopy. Envelopes from immature oocytes at the stage of beginning yolk deposition were compared with those from mature oocytes. Envelopes from the immature stage had "pores" whose annuli contained more material and showed central globules in the center much more frequently than envelopes from mature eggs. Annuli and central globules had similar appearance and fixation properties, suggesting similar chemical composition. After fixation with KMnO₄, residual densities suggested that "pore" diaphragms are much more variable in thickness or composition in the younger stages. Envelopes of the immature oocytes had about 40 per cent more "pores" per unit area than mature envelopes. In crowding together, the "pores" tended to assume geometrical packing arrays in the young envelope, showing minimum center-to-center spacings of about 1530 A. Since the actual discontinuities in the membranes of the envelope are only about 950 A in diameter, this minimum distance of approach suggests that adjacent formations of the nuclear surface are associated with "pore" structure and perhaps set their limiting spacing distances. If this is true, then it can be deduced that "pore"-associated structures of the nuclear surface are probably circular in outline and about 1500 A in diameter. Isotopically labeled lysine was administered to intact, growing oocytes for 1 to 4 hours and the envelopes were subsequently isolated and fixed. Autoradiography of entire envelopes showed little or no incorporation of lysine into proteins, as compared with small fragments from other parts of the cell of roughly comparable mass. It was concluded that the isolated envelope, as seen in the electron micrographs, does not synthesize or turn over lysine-containing protein at a high rate.     

Fusion between Sendai virus envelopes and biological membranes. The use of fluorescent probes for quantitative estimation of virus-membrane fusion

The fluorescent probes, N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine and lissamine-rhodamine-B-sulfonylphosphatidylethanolamine, were inserted at the appropriate surface density into membranes of reconstituted Sendai virus envelopes, thus allowing transfer of energy between the fluorescent probes. In addition, only the fluorescent molecule N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine was inserted into the viral envelopes, resulting in self-quenching. Incubation of fluorescent, reconstituted Sendai virus envelopes with human erythrocyte ghosts resulted in either reduction in the efficiency of energy transfer or in fluorescence dequenching. No reduction in the efficiency of energy transfer or fluorescence dequenching was observed when fluorescent, reconstituted Sendai virus envelopes were incubated with glutaraldehyde-fixed or desialized human erythrocyte ghosts. Similarly, no change in the fluorescence value was observed when nonfusogenic, reconstituted Sendai virus envelopes were incubated with human erythrocyte ghosts. These results clearly show that reduction in the efficiency of energy transfer or dequenching is due to virus-membrane fusion and not to lipid-lipid exchange. Incubation of reconstituted Sendai virus envelopes, carrying inserted N-4-nitrobenzo-2-oxa-1,3-diazolephosphatidylethanolamine, with cultured cells also resulted in a significant and measurable dequenching. However, incubation of nonfusogenic, fluorescent reconstituted Sendai virus envelopes with hepatoma tissue culture cells also resulted in fluorescent dequenching, the degree of which was about 50% of that observed with fusogenic, fluorescent reconstituted viral envelopes. It is therefore possible that, in addition to virus-membrane fusion, endocytosis of fluorescent viral envelopes results in fluorescence dequenching as well.     

Controlled production of spheroplasts in the yeast Nadsonia elongata

Summary Yeast cells of Nadsonia elongata were cultivated in such a way that simultaneously with enzymatic lysis of the cell wall a partial synthesis of cell wall components was taking place. After the initial period of cultivation, which lasted about 10 h and during which the morphology of cells remained unchanged when compared to controls, the cells were transformed into prospheroplasts. The prospheroplasts were larger than the control cells and, though they enlarged in volume in distilled water, they still retained the shape of the original cells. However, some changes were found in the ultrastructure of the cell walls of prospheroplasts in comparison with that of the cell walls of intact cells: while in yeast cells the surface was smooth, in prospheroplasts the fibrillar network was revealed as a result of the removal of the amorphous component; the gradual disappearance of the outer cell wall layer and a swelling of the remaining cell wall fragment were seen in ultrathin sections. After about 20-h cultivation the prospheroplasts were transformed into spheroplasts. The spheroplasts were osmotically fragile, and did not retain the shape of the yeast cell, even in isoosmotic environment. On the surface of spheroplasts only the fibrillar network composed of separate fibrils was seen. The spheroplasts were the final stage of yeast cell transformation under the conditions employed in the present study. Under the mentioned conditions true protoplasts are never formed. However, if the synthesis of cell wall components could not take place simultaneously with the lysis of the cell wall, the cells were transformed to protoplasts.     

FINE STRUCTURE AND ORGANELLE ASSOCIATIONS IN BROWN ALGAE

The structural interrelationships among several membrane systems in the cells of brown algae have been examined by electron microscopy. In the brown algae the chloroplasts are surrounded by two envelopes, the outer of which in some cases is continuous with the nuclear envelope. The pyrenoid, when present, protrudes from the chloroplast, is also surrounded by the two chloroplast envelopes, and, in addition, is capped by a third dilated envelope or "pyrenoid sac." The regular apposition of the membranes around the pyrenoid contrasts with their looser appearance over the remainder of the chloroplast. The Golgi apparatus is closely associated with the nuclear envelope in all brown algae examined, but in the Fucales this association may extend to portions of the cytoplasmic endoplasmic reticulum as well. Evidence is presented for the derivation of vesicles, characteristic of those found in the formative region of the Golgi apparatus, from portions of the underlying nuclear envelope. The possibility that a structural channeling system for carbohydrate reserves and secretory precursors may be present in brown algae is considered. Other features of the brown algal cell, such as crystal-containing bodies, the variety of darkly staining vacuoles, centrioles, and mitochondria, are examined briefly, and compared with similar structures in other plant cells.     

Hatching envelope formation in shrimp (Sicyonia ingentis) ova: Origin and sequential exocytosis of cortical vesicles

The ova of Sicyonia ingentis lack cortical vesicles at the time of spawning. Within 30 min post-spawning, two populations of cortical vesicles are organized in the ooplasm which, during cortical vesicle exocytosis (cortical reaction), successively release two morphologically different exudates. The first type of cortical vesicles (dense vesicles) appears to be derived from the Golgi complexes after spawning. The second type (the ring vesicles) is formed by the fusion of asternal elements which contain loosely packed ring-shaped structures that are present in the unactivated ova. During exocytosis of the dense vesicles an electron dense material is released which coalesces with the surface coat of the ovum to form a thin hatching envelope which eventually lifts from the ovum's surface. Subsequent to the formation of the thin hatching envelope, the ring vesicles undergo exocytosis resulting in an accumulation of ring-shaped structures in the perivitelline space. These structures coalesce and form an electron translucent layer on the inner surface of the thin elevated envelope to form the thickened hatching envelope. The formation of the cortical vesicles, their exocytosis and the elaboration of the hatching envelope are normally completed within 40-45 min after spawning.     

Development of the Embryonic Envelopes of Mesocestoides lineatus (Cestoda: Cyclophyllidea)

Summary

Transmission electron microscopy revealed that the eggs of Mesocestoides lineatus consisted of an oncosphere larva surrounded by various coverings. The outermost of these was the embryonic capsule, which appeared as a thin electron-dense membranous sac. The capsule enclosed inner and outer embryonic envelopes, each of which was syncytial and apparently formed from embryonic blastomeres. The envelopes became increasingly vesiculated during embryogenesis, and were attached to each other by desmosomes by the time the larva was fully formed. An electron-dense intracellular embryophore was produced by the inner envelope; it first appeared under the distal plasma membrane as a series of blocks, which grew and fused to form a thick unbroken layer. Early in development, the proximal plasma membrane of the inner envelope was connected to the larval epithelium by a multilaminate membrane complex that was ultrastructurally similar to a continuous junction. At the end of embryogenesis, this appeared to detach from its formative cells on both sides to form the distinctive oncospheral membrane. Several eggs were bound together in clusters by a cluster capsule that was ultrastructurally identical to the individual embryonic capsules. This type of egg packaging has not been described previously for any cestode. Both the cluster and individual capsules broke down by the end of embryogenesis.     

Activation, stimulation and uptake of bacterial ghosts in antigen presenting cells

Bacterial ghosts have been shown to be an innovative system to prepare vaccines of various bacteria with all features of the intact bacterial cell envelopes, especially all antigenic epitopes, but also to target recombinant proteins inserted in the cell envelopes of the ghost preparations to specific antigen presenting cells. To investigate the activation of the antigen presenting cell by bacterial ghosts in more detail we studied the uptake of bacterial ghosts in dendritic porcine cells and RAW macrophages and the induction of inflammatory mediators or mediators directing the immune response in THP-1 human macrophage cell line. The synthesis of inflammatory macrophage mediators such as TNFalpha in the THP1 cell line was stimulated by a hundred-fold higher dose of ghosts from Vibrio cholerae than the corresponding LPS using ELISA-analysis. These results confirm in vivo experiments indicating no toxic effects of ghosts in rabbits even after intravenous administration in doses stimulating significant humoral responses. We were also able to see a significant activation of IL-12 indicated by the analysis of IL-12(p70) synthesis and IL-12(p40) mRNA accumulation. This interleukine is of special importance in the activation of cellular TH1 immune responses. A rapid uptake of bacterial ghosts in macrophages within 10-30 min could be confirmed by electron microscopy. As antigen presentation is especially effective in porcine dendritic cells (DC) and even a low capacity of antigen uptake is sufficient for an induction of immune responses we investigated uptake and activation of bacterial ghosts by DC. DC are known to be phagocytic in specific immature stages. We found a significant uptake of bacterial ghosts from Actinobacillus pleuropneumoniae (App) and V. cholerae conjugated with FITC (fluorescinisothiocyanate) within 2 h. These data suggest that bacterial ghosts effectively stimulate monocytes and macrophages for the induction of TH1 directed immune responses and dendritic cells treated with bacterial ghosts may serve as a promising vehicle for active immunization and immunotherapy in situ.     

Detection of dense intra- and perinuclear 10 nm filament systems by whole mount and embedment-free electron microscopy in several species of the green algal order Dasycladales

Summary In contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with M_rs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the -IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.Dedicated to Professor Sir Henry Harris on the occasion of his 70th birthday