Immunocytochemical study of the endocrine pancreas in the grey kangaroo (Macropus fuliginosus)

Summary The endocrine pancreas of the grey kangaroo,Macropus fuliginosus, was investigated by means of immunocytochemistry using the PAP method on the same section at the light- and electron-microscopic levels. Semithin plastic sections were stained individually with primary antibodies for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), and then photographed. Sections were osmicated, re-embedded in BEEM capsules, and ultrathin sections made and examined. The same labelled cells as in the semithin sections were localised in the thin sections, photographs taken and the morphology of secretory granules studied. The insulin cells were pleomorphic; their secretory granules displayed an electron-dense core surrounded by an empty halo. The glucagon cells possessed granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells had larger, less dense secretory granules. The PP cells showed small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborted by correlated immunocytochemical data at the light-and electron-microscopic levels.     

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

We have analysed the intracellular localisation of phytochrome in oat coleoptile cells by electron microscopy and confirm and extend light-microscopical findings of previous authors. We used indirect immuno-labeling with polyclonal antibodies against 60-KDa phytochrome from etiolated oat seedlings, and a gold-coupled second antibody, on ultrathin sections of LR-white-embedded material. In dark-grown seedlings, phytochrome-labeling is distributed diffusely throughout the cytoplasm. Organelles and membranes are not labeled. After photoconversion of the red-absorbing form of phytochrome to the far-red absorbing form (Pfr) (5-min red light; 660 nm), the label is sequestered uniquely in electron-dense areas within the cytoplasm. These areas are irregularly shaped, are often located in the vicinity of the vacuole, are not surrounded by a membrane, exclude cellular organelles and ribosomes and are not found in dark-grown material; an immediate 5-min farred light pulse after the red light does not cause these structures to disappear. After a dark period of 3–4 h following red-light irradiation, these electron-dense structures disappear together with any specific labeling. We suggest a Pfr-induced aggregation of an unknown, phytochrome-binding protein or proteins.Abbreviations Pr and Pfr phytochrome in its red and far-red absorbing form, respectively     

The chitin-glucan complex ofSaccharomyces cerevisiae

Differentiation of the cell wall ofSaccharomyces cerevisiae at the site of the future bud was followed. A lentil-like structure originates on the inner side of the cell wall during the first phase. At the same time, an electron-dense layer occurs at the boundary between the inner layer of the cell wall and the lentil-like structure. During the second phase granular material is accumulated at the lower side of the lentil-like structure. During the third phase the lentil-like structure is split apart due to proliferation of the granular material resulting in formation of the base of the encircling region. The marked electron-dense layer observed from the first phase is attached to the surface of the encircling region during differentiation of the latter. During the budding proper the outer layers of the cell wall protrude and the end of the encircling region, together with the adjacent electron-dense layer, acquire their definitive appearance of rings, observed as marked electron-transparent and electron-dense tears on ultrathin sections.     

Boolarra virus: Ultrastructure of intracytoplasmic virus formation in cultured Drosophila cells

Boolarra virus (BoV) is a Nodavirus isolated from Oncopera intricoides. Drosophila cell lines 1 (D1) and 2 (D2) were infected with virus and the progession of infection was followed in ultrathin sections viewed by the electron microscope. Viral morphogenesis was restricted to the cytoplasm. Virogenic stroma condensed to electron-dense areas in which were embedded electron-lucent and electron-dense particles. picornaviruslike particles 30 nm in diameter were found in membranous channels and paracrystalline arrays and were scattered throughout the cytoplasm of cells. Virus was released from cisternae and tracts which extended to the cells' periphery. Local disruption of cell membranes also released particles and aggregates of virus into the environment.     

Electron microscopic investigation of the hydrogen-oxidizing acetate-forming anaerobic bacterium Acetobacterium woodii

Acetobacterium woodii is a Gram-positive anaerobic nonsporeforming bacterium able to grow on H₂ and CO₂ as sole sources of energy. The product of fermentation is acetic acid. Fine structural analysis showed rod-shaped flagellated cells, and coccoid cells without flagella arranged predominantly in pairs and chains. The cell wall was found to be composed of three layers. The cell surface exhibited a periodic array of particles consisting of subunits. The cytoplasmic membrane showed particles either either in random distribution or in a hexagonal pattern. Intracytoplasmic membranes were rarely observed, whereas inclusion bodies of varying shapes, predominantly in an uncommon disc-shape, could frequently be observed. Their content was dissolved in ultrathin sections indicating hydrophobic nature.     

Ca2+-binding sites and Ca2+-ATPase activity in barley root tip cells

Summary Different cytochemical methods were employed to demonstrate the existence of Ca²⁺-binding sites (Ca²⁺-bs) at the membranes of barley root tip cells, involving addition of CaCl₂ (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca²⁺-dependent ATPase activity. Addition of 10 mM CaCl₂ to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl₂ electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl₂) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca²⁺-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion.     

Electron microscopic localization of concanavalin A receptor sites in pollen surface material after fixation with glutaraldehyde-cetylpyridinium chloride

Pollen from birch trees (Betula pendula) was fixed in glutaraldehyde containing 0.5% cetylpyridinium chloride (CPC), incubated with concanavalin A (Con A)-ferritin, postfixed in osmium, dehydrated, and embedded in Epon. On ultrathin sections, ferritin particles were observed closely associated with the electron-dense material precipitated by CPC on the surface of the pollen grains. Controls for CPC, which were fixed in glutaraldehyde alone, showed no electron-dense material on the surface. In controls for Con A, which were incubated in Con A-ferritin in the presence of the inhibitory sugar (alpha-methyl-D-mannopyranoside), no ferritin particles were observed. The above-described procedure thus allows the localization of sugar residues in highly soluble pollen wall glycoproteins.     

Electron microscopy of infection of nematodes byDactylaria haptotyla

Infection of nematodes byDactylaria haptotyla, a nematode-trapping hyphomycete, was studied by electron microscopy. The cytoplasm of the adhesive knob in the fungus contained a number of electron-dense, membrane-bound vesicles, 0.2–0.5 µm in diam. The vesicles were rarely seen in the stalk cell or vegetative cell cytoplasm. When the adhesive knob came into contact with the nematode's cuticle, it secreted an adhesive which was seen in ultrathin sections between the knob and the cuticle as an amorphous mass. At the same time, electron-dense vesicles in the cytoplasm were reduced in number and many small vacuoles developed. Soon after capture of a nematode, the cell wall of the adhesive knob became obscure at the prospective site of penetration, where a vesicle, 0.7 µm in diam, was found in serial thin sections of the knob's cytoplasm. At the site facing the vesicle, the peripheral part of the nematode's cell exhibited a high electron density. The vesicle, which appeared to be derived from smaller electron-dense vesicles coalesced with each other, released its enzymic contents toward the captured nematodes before penetration by the fungus.     

Use of protein A-coated colloidal gold particles for immunoelectronmicroscopic localization of ACTH on ultrathin sections

Summary ACTH was localized in dissociated porcine adenohypophysial cells using a novel indirect EM immunocytochemical technique. Incubation of ultrathin resin sections in anti-ACTH was followed by incubation with protein A-coated colloidal gold particles. Protein A binds specifically to the Fc part of the IgG molecule, and thus the ACTH-containing secretory granules became labelled with electron-dense gold particles. With this method, the dissociated porcine ACTH cell was identified as containing numerous round or ovoid 170–300 nm, secretory granules.     

Pseudomonas bacteriophage phi KZ contains an inner body in its capsid

Electron microscopical examination of the new virulent bacteriophage phi KZ, specific for Pseudomonas aeruginosa, has revealed an unusual structure in its capsid. In the center of the phage head is a cylinder of low electron density ("inner body"), surrounded by fibrous material which is packed around the inner body in a spoollike manner. The inner body itself has a springlike appearance. These structures disappear after adsorption of phage particles to bacteria. Various morphological forms, which can be interpreted as intermediate steps in phi KZ DNA condensation, have been seen in ultrathin sections of phi KZ-infected cells.     

Ruthenium Red Stain Able Surface Layer on Lung Alveolar Cells; Electron Microscopic Interpretation

Luft's ruthenium red (RR) method was applied to lung tissue. Small blocks of mouse lung were fixed for 1 hr with 1.2% glutaraldehyde at 0-4 C, buffered with 0.067 M cacodylate, pH 7.3 and containing RR, 1 mg/ml. Following fixation, lung blocks were immersed in 0.15 M cacodylate for 10 min and postfixed for 3 hr at room temperature with 2% OsO₄ buffered with 0.067 M cacodylate, pH 7.3, and containing RR, 1 mg/ml. Blocks were dehydrated with ethanol, embedded in Araldite, and ultrathin sections treated with uranyl acetate and lead citrate solutions to enhance contrast of cell structures. Electron micrographs revealed an electron-dense layer coating the exposed surfaces of alveolar cells. This layer corresponded in location and appearance to that observed by other investigators who used colloidal iron techniques.     

Use of protein A-coated colloidal gold particles for immunoelectronmicroscopic localization of ACTH on ultrathin sections.

ACTH was localized in dissociated porcine adenohypophysial cells using a novel indirect EM immunocytochemical technique. Incubation of ultrathin resin sections in anti-ACTH was followed by incubation with protein A-coated colloidal gold particles. Protein A binds specifically to the Fc part of the IgG molecule, and thus the ACTH-containing secretory granules became labelled with electron-dense gold particles. With this method, the dissociated porcine ACTH cells was identified as containing numerous round or ovoid 170--300 nm secretory granules.     

Immunological detection and localization of a calsequestrin-like protein in red beet and cucumber cells

Summary Calsequestrin is a calcium binding protein present in the sarcoplasmic reticulum (SR) of animal muscle cells and is thought to be essential for the rapid uptake and release of Ca²⁺, and thus for the regulation of Ca²⁺-dependent cellular functions. Higher plant cells of red beet (Beta vulgaris L.) and cucumber (Cucumis sativus L.) contain a polypeptide of about M_r 55000 that cross-reacts with a monoclonal antibody raised against calsequestrin from rabbit skeletal muscle SR. In beet this protein changes its apparent molecular weight with pH as indicated in Western immunoblotting. Although this protein bound calcium it was not the dominant calcium-binding protein in red beet. Washing of beet root tissue leads to a slight increase of this polypeptide in microsomal fractions as indicated by immunoblotting. After immunoblotting to partially purified cell membrane fractions this polypeptide appeared to be predominantly associated with endoplasmic reticulum-enriched fractions. Immunogold labelling of ultrathin sections of cucumber hypocotyl using the anti-calsequestrin antibody showed that gold particles were very largely confined to the cytosol and often in close proximity to the ER. Clusters of up to nine gold particles were observed, often over small vesicular areas, as observed in some animal tissues. These results indicate that red beet and cucumber cells contain a protein which may be related to animal calsequestrin. It appears to be associated with the ER and could be involved in cellular calcium regulation.     

THE PARASPORAL BODY OF BACILLUS LATEROSPORUS LAUBACH

On sporulation the slender vegetative rods swell and form larger spindle-shaped cells in which the spores are formed. When the spores mature they lie in a lateral position cradled in canoe-shaped parasporal bodies which are highly basophilic and can be differentiated from the surrounding vegetative cell cytoplasm with dilute basic dyes. On completion of sporulation the vegetative cell protoplasm and the cell wall lyse, leaving the spore cradled in its parasporal body. This attachment continues indefinitely on the usual culture medium and even persists after the spores have germinated. In thin sections of sporing cells the bodies are differentiated from the cell protoplasm by differences in structure. Whereas the protoplasm has a granular appearance, in both longitudinal and cross-sections the parasporal body comprises electron-dense lamellae running parallel with the membranes of the spore coat and less electron-dense material in the interstices of the lamellae. The inner surface of the body is contiguous with that of the spore coat as if it were part of the spore, rather than a separate body attached to the spore. The staining reactions of the parasporal body are not consistent with those of any substance described in bacteria. With Giemsa the bodies stain like chromatin, but the Feulgen reaction indicates that they do not contain the requisite nucleic acid. With an aqueous solution of toluidine blue they stain metachromatically, but with an acidified solution the results are variable. Neisser's stain for polyphosphate is negative. The basophilic substance is removed from the body with some organic solvents. This basophilic substance has not been specifically identified with any material seen in ultrathin sections, but it is suggested that it might be the less electron-dense material in the interstices of the lamellar structure. In contrast to the spore coat of B. laterosporus, those of its two relatives B. brevis and B. circulans take up basic stain like the parasporal body. Thin spore sections of these species have shown that the walls are thicker than those surrounding the spores of B. laterosporus, and it is suggested that the outer stainable layer of brevis and circulans spores is an accessory coat which in laterosporus may have been deformed to give a parasporal body.     

Visualization of proteins in intact cells with a clonable tag for electron microscopy

Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.     

NaHS对NaHCO3胁迫下黑籽南瓜种子萌发及生理特性的影响

以黑籽南瓜(Cucurbita ficifolia)种子为试材, 研究了外施不同浓度的NaHS对NaHCO₃胁迫下种子萌发及生理特性的影响。结果表明, NaHCO₃胁迫显著抑制了黑籽南瓜种子的发芽率、胚轴长和胚根长, 降低了种子萌发过程中的可溶性糖含量, 抑制了α-淀粉酶、β-淀粉酶、SOD及POD活性。而外施不同浓度的NaHS显著促进了NaHCO₃胁迫下黑籽南瓜萌发种子胚轴和胚根的生长, 提高了可溶性糖含量及α-淀粉酶、β-淀粉酶、SOD和POD活性, 降低了MDA含量; 外施其它盐类(Na₂S、Na₂SO4、NaHSO₄和NaHSO₃)及不同pH值(pH5.8–7.8)的Na₂HPO₄-NaH₂PO₄缓冲液对NaHCO₃胁迫下黑籽南瓜种子的萌发则无影响。外施NaHS可有效缓解NaHCO₃胁迫对黑籽南瓜种子萌发的抑制作用, 其缓解效应可能与其释放的H₂S有关。     

Immunocytochemical localization of reserve protein in the endoplasmic reticulum of developing bean (Phaseolus vulgaris) cotyledons

The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at-80° C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 m. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G     

The role of acidification in gibberellic acid- and fusicoccin-induced elongation growth of lettuce hypocotyl sections

The roles of gibberellic acid (GA₃) and fusicoccin (FC) in the elongation growth and acidification of the medium by excised hypocotyl sections of lettuce (Lactuca sativa L.) were investigated. Hypocotyl sections incubated in buffer without GA₃ elongate optimally at pH 4.0–4.25 while sections incubated with GA₃ show the same growth between pH 4.25 and 6.0. Preincubation of sections at pH 6.0 for 6 h does not affect the subsequent elongation response to acidic medium (pH 4.25); however, the sections become refractory to further acid treatment after their initial burst of growth in response to pH 4.25. Sections made refractory to acid are responsive to GA₃ application, however, and the rate of growth in response to GA₃ of sections pretreated for 6 h at pH 4.25 is 85% of that of sections pretreated at pH 6.0. Although preincubation of sections for 48 h in medium at pH 6.0 abolishes the GA₃ response, it does not affect the response to buffer at pH 4.25. FC stimulates elongation growth in letuce hypocotyls at an optimal concentration of 1 M, and pretreatment of sections at pH 4.25 does not affect this elongation response. Although both GA₃ and FC increase elongation of the section, neither causes appreciable acidification of the medium. Addition of KCl or NaCl to FC-treated sections causes rapid medium acidification but addition of salts to GA₃-treated tissue does not cause acidification. Abrasion of the hypocotyl to remove the cuticle does not enhance acidification of the medium by the sections nor deos it affect elongation of the sections in response to GA₃ or FC. Medium acidification by the sections is not a passive process since it is abolished both by low temperature (2° C) and metabolic inhibitors (carbonyl cyanide-m-chlorophenyl-hydrazone, azide). The acidification of the medium by barley (Hordeum vulgare L.) roots in response to FC is also dependent on the presence of KCl. We conclude that the acid-growth hypothesis does not explain GA₃- or FC-induced elongation in lettuce hypocotyls.Abbreviations FC tusicoccin - GA₃ gibberellic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - MES 2-(N-morpholino)ethanesulphonic acid - Tris tris-(hydroxymethyl)aminomethane     

ELECTRON MICROSCOPY OF WOOD OF CALLIXYLON AND CORDAITES

Replicas and ultrathin sections of the wood of two Paleozoic genera, Callixylon and Cordaites, were examined with the electron microscope. The pattern of wall layering of Callixylon closely resembles that of extant plants. An electron-dense compound middle lamella markedly thickened at the corners of cells, a thin, electron-transparent S₁ layer of the secondary wall, and a thick, electron-dense, partially decayed S₂ layer of the secondary wall are evident in transverse sections of tracheids. No S₃ layer seems to be present. The structure of the bordered pit-pairs of Callixylon is described in detail. The slitlike outer pit apertures are conspicuously narrower and shorter than the inner pit apertures. Both sections and replicas of the bordered pit-pairs display pit membranes lacking tori. Microfibrillar structure is obscure in both sections and replicas of Callixylon wood. Replicas of the bordered pits of Cordaites wood are very similar to those of Callixylon. Pit membranes lack tori, and microfibrillar structure is not very discernible. Knowledge about the evolution of the torus is summarized. It is postulated that the type of pit membrane of Callixylon and Cordaites, which is very homogeneous in structure and lacks a torus, represents a primitive condition among gymnosperms from which structurally more complex pit membranes and the torus later evolved.     

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.