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Photosomes are the characteristic organelles of the luminous epithelium in the elytral appendages of polynoïd annelids. They are paracrystals of endoplasmic reticulum and emit a flash of bioluminescence in response to stimulation. The series of flashes in response to repetitive stimulation begins with a period of facilitation because the number of reacting photosomes increases in each photogenic cell. Reacting photosomes are coupled to the plasma membrane by dyad junctions which are established under stimulation and dedifferentiate in the resting system. The calcium influx of an action potential propagated through the conducting elytral epithelium triggers the luminous reaction. This reaction is based on a membrane photoprotein, polynoidin, which is specifically triggered by superoxide radicals. These oxy radicals result from the oxydation of riboflavin, which is present in a compartment of the photosomes. Polynoidin proved to be an interesting probe in the detection of superoxide radicals produced by activated white blood cells. Its potential applications are discussed.

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As compared to classical chemical fixation, the physical immobilization of ultrastructures by fast-freeze fixation (FFF) and the subsequent exchange of water in its solid state by freeze substitution (FS) improve the preparation procedure for immunogold labeling (IGL). FFF-FS results in a morphological preservation of unchallenged quality, as well as in a better preservation of antigenic reactivity, thus allowing remarkable precision of labeling on sections. However, FFF, particularly over a cooled metal plate, requires a heavy and expensive machine. It is not suitable for all biological specimens and in the best conditions, which remain difficult to standardize, the thickness of the well-preserved portion of the specimen does not exceed a few microns for compact tissues, and exceptionally 30-40 microns for isolated cells. The FS procedure is long and must be adjusted empirically for every new specimen and antigenic detection. The preservation of a given antigen's reactivity in the presence of fixative agents and embedding resins remains unpredictable. The action of fixative agents is different and milder in FS than when they are used classically in chemical fixation. By chance, one of the best FS procedures for the preservation of both ultrastructure and antigenicity appears to be by using acetone alone, together with a molecular sieve to improve the water exchange process. A large choice of embedding resins usually allows us to find a compromise between ultrastructural and antigenic preservation.

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Elastase is a potent proteolytic enzyme found within human neutrophil primary granules. Its major inhibitor in the serum is alpha 1-antitrypsin, a protein that is synthesized by hepatocytes but which has recently also been shown to be synthesized by circulating neutrophils. The authors have therefore carried out an immunocytochemical study at the light microscopic and ultrastructural level to determine the intracellular localization of alpha 1-antitrypsin. Double labeling with colloidal gold showed that alpha 1-antitrypsin is localized at the same site as neutrophil elastase, i.e., within primary granules. Secondary granules (detected by labeling for lactoferrin) were unstained for alpha 1-antitrypsin. Elastase and its major inhibitor therefore coexist within the same granule population within human neutrophils. Some difference in their intraorganelle distribution existed at the ultrastructural level (in that elastase tended to be localized at the periphery of the granules whereas alpha 1-antitrypsin was usually diffusely present in the matrix of the granules), but further studies are required to determine whether the two molecules are already complexed with each other within the neutrophil.

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Using the luminescent protein polynoidin, present in the bioluminescent system isolated from the marine annelid Harmothoe lunulata, we have developed a new method to measure, specifically, superoxide anion (O2-) released by macrophages or neutrophils. A small quantity of an aqueous crude extract of polynoidin is used to detect O2- released by stimulated cells. Light emission is linearly dependent on the number of cells over a wide range (10(3) to 10(7) cells), and the assay is thus more sensitive than either luminol or ferricytochrome c reduction. Luminescence is enhanced 20% by mannitol, 80% by catalase, and is totally quenched by superoxide dismutase. For the same number of cells, neutrophils showed a threefold higher release of O2- and a twofold faster first-order light decay than stimulated macrophages, in accordance with data obtained by other methods.

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We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.

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Isolated elytra of polynoid worms emit a flash of bioluminescence when stimulated by an electric shock. With repeated stimulation, hundreds of flashes can be elicited which, in typical series, exhibit large and progressive variations. The amount of luminescence emitted by each flash first increases during a period of facilitation and then decreases exponentially during a longer period of decay. Through a microscope and image intensifier, the activity of individual microsources or photosomes was observed, using their fluorescence as a natural probe, in that its intensity is a function of the amount of luminescence previously emitted. Sequential observation showed a progressive and basically intracellular recruitment that correlated with facilitation. Facilitation and/or recruitment depended on the frequency of the stimulation. Recruitment proceeded among the photosomes of each photocyte, beginning with those of the cell periphery and progressing to those of the center. When the repetitive stimulation was interrupted and then resumed, the refacilitation was a function of the duration of the pause, and the pathway of recruitment duplicated that of the preceding sequence. It therefore appears that, within a given cell, individual photosomes can be either coupled and respond to stimulation or uncoupled and quiescent, that the coupled state has a basic lifetime of about 1 s which can be lengthened by reinforcement, and that this state must be established in a matter of milliseconds as a result of the stimulation. In preparing an increased response to a forthcoming stimulation, coupling acts as a short-term memory.

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In the bioluminescent system of the scale worm, the facilitation of the successive flashes is correlated with the progressive recruitment, in each photogenic cell, of new units of activity, the photosomes. To characterize morphologically the coupled state of the photosomes, known to decouple within seconds at rest, fast-freeze fixation was applied to stimulated and nonstimulated elytra and followed by substitution with OsO4 in acetone. The results showed striking differences. Photosomes were surrounded by a new type of smooth endoplasmic reticulum (ER) called intermediate endoplasmic reticulum (IER). In nonstimulated elytra, the IER was most often unattached in the cytoplasm. After stimulation, the IER was connected to large terminal saccules that formed dyad junctions with the plasma membrane. Most of these junctional complexes were symmetrical (triads) and occurred in front of narrow extracellular spaces. These spaces were either constitutive, like invaginations or clefts along adjacent cells and adjacent pouches, or resulted from the pairing of long pseudopods which expanded into a wide extracellular compartment and twisted together in a dynamic process. In that the junctional complexes developed progressively under repeated stimulation and coupled more and more photosomes, they must represent a route constituted by the ER for the propagation of internal conduction. The dynamics of coupling involve membrane growth, recognition, and transformation on a surprisingly large scale and in a surprisingly short time.

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To characterize the microsources of bioluminescent activity in the dinoflagellate Gonyaulax polyedra, an immunogold labeling method using a polyclonal antiluciferase was combined with fast-freeze fixation and freeze substitution. The quality of the preservation and the specificity of the labeling were greatly improved compared to earlier results with chemical fixation. Two organelles were specifically labeled: cytoplasmic dense bodies with a finely vermiculate texture, and mature trichocysts, labeled in the space between the shaft and the membrane. The available evidence indicates that the dense bodies are the light-emitting microsources observed in vivo. The dense bodies appear to originate in the Golgi area as cytoplasmic densifications and, while migrating peripherally, come into contact with the vacuolar membrane. Mature organelles protrude and hang like drops in the vacuolar space, linked by narrow necks to the cytoplasm. These structural relationships, not previously apparent with glutaraldehyde fixation, suggest how bioluminescent flashes can be elicited by a proton influx from a triggering action potential propagated along the vacuolar membrane. Similar dense bodies were labeled in the active particulate biochemical fraction (the scintillons), where they were completely membrane bound, as expected if their necks were broken and resealed during extraction. The significance of the trichocyst reactivity remains enigmatic. Both organelles were labeled with affinity-purified antibody, which makes it unlikely that the trichocyst labeling is due to a second antibody of different specificity. But trichocysts are not bioluminescent; the cross-reacting material could be luciferase present in this compartment for some other reason, or a different protein carrying similar antigenic epitopes.

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A polyclonal antibody directed against the luciferase of the luminous dinoflagellate Gonyaulax polyedra labels both dense vesicles and trichocyst sheaths, as visualized in the electron microscope after treatment of antibody-reacted sections with an immunogold probe. Because of their similar size, shape and localization, the dense vesicles seen with the electron microscope are postulated to correspond to autofluorescent particles seen with the fluorescent microscope, which are known to be the origin of bioluminescent flashes in this alga. The explanation for the trichocyst sheath-specific labeling is less evident. The possibility that a second antibody of different specificity is involved has not been excluded but seems unlikely. Alternatively, it could be due to a different but antigenically cross-reacting protein. But the possibility that luciferase itself occurs in two different organelles is intriguing and consistent with previous biochemical studies of cell extracts.

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Luminous cells of polynoid worm elytra have been examined by methods of electron microscopy, with special attention focused on the fine structure of photogenic grains. These cells send apical prolongations into the mid-part of the elytra. The plasma membrane is very sinuous, and a special kind of desmosome links two portions of the same membrane. In addition to all the organelles which can be found in nonluminescent epithelial cells of the elytra, numerous photogenic grains are contained in their cytoplasm. These grains are composed of undulating microtubules measuring 200 A in diameter; their disposition in the grain is highly regular, and the grains appear as paracrystals. At the borders of the grains, the walls of the microtubules are often in continuity with those of the endoplasmic reticulum and with the external membrane of the nuclear envelope. Because of this fact, the microtubules of the grains may be considered a cytoplasmic organelle, representing a specialized form of the endoplasmic reticulum. The microtubules permit the repartition, inside and outside their walls, of two different products, one being forty-three times more abundant than the other; thus, the contact surface, in comparison to the volume, is greatly increased. The induction of the luminous reaction by change in the permeability of the microtubule walls, allowing contact between the two substances, is suggested as a working hypothesis. There is an evolution of the grains along the axis of the photocytes. The grains are often surrounded by progressively increasing amounts of glycogen. Their paracrystalline disposition is altered at the apex of the luminous cells.

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