Membranous Whorls Research Articles

Pathological alterations typical of human Tay-Sachs disease, in the retina of a deep-sea fish.

Micrographs of retinas from the deep-sea fish Cataetyx laticeps revealed visual cells containing membranous whorls in the ellipsoids of the inner segments resulting from stretching and modifications of the mitochondria membranes and their cristae. These pathological structures seem to be homologous to the whorls observed in retinas of human carriers of Tay-Sachs disease. This disease, a genetic disorder, is found in humans and some mammals. Our findings in fish suggest that the gene responsible can be found throughout the vertebrate evolutionary tree, possibly dormant in most taxa.

Proteasome inhibitors block intracellular growth and replication of Toxoplasma gondii

Lactacystin, a specific inhibitor of proteasomes in eukaryotic cells, did not block parasite entry or the establishment of the parasitophorous vacuole, but did inhibit parasite growth and daughter cell budding, as well as DNA synthesis. Two other proteasome inhibitors, MG-132 and proteasome inhibitor 1, also blocked parasite growth and intracellular development. Adding lactacystin to established, dividing parasites, rapidly blocked parasite growth and daughter cell budding at all stages in the process. Pre-treating host cells with lactacystin did not block parasite entry or development. Moreover, under the conditions used, the host cells appeared not to be adversely affected indicating that host cell proteasome activity was not essential for parasite entry or development. Concomitant with these effects on parasite growth and division were morphological changes in the parasite including the appearance of whorls of ER-derived membranes presumably related to the failure to breakdown misfolded proteins. These changes were specific to lactacystin and were not seen in parasites treated with other protease inhibitors. Although the ER-derived structures resembled autophagic bodies, similar structures could not be induced by serum starvation nor did the membranous whorls acidify or undergo morphological changes consistent with autophagosomal maturation. These results highlight the possible role of proteasome activity in Toxoplasma in intracellular development and the regulation of parasite replication. However, how the dividing parasite recycles its organelles and the functional relationship between any lysosomal autophagic pathway and proteasomes in the parasite remains unresolved.

Mutant torsinA, responsible for early-onset torsion dystonia, forms membrane inclusions in cultured neural cells

Early-onset torsion dystonia is a hereditary movement disorder thought to be caused by decreased release of dopamine into the basal ganglia, without apparent neuronal degeneration. Recent cloning of the gene responsible for this disease, TOR1A (DYT1), identified the encoded protein, torsinA, as a member of the AAA+ superfamily of chaperone proteins and revealed highest levels of expression in dopaminergic neurons in human brain. Most cases of this disease are caused by a deletion of one glutamic acid residue in the C-terminal region of the protein. Antibodies generated against torsinA revealed expression of a predominant immunoreactive protein species similar to the predicted size of 37.8 kDa in neural, glial and fibroblastic lines by western blot analysis. This protein is N-glycosylated with high mannose content and not, apparently, phosphoryl-ated. Overexpression of torsinA in mouse neural CAD cells followed by immunocytochemistry, revealed a dramatically different pattern of distribution for wild-type and mutant forms of the protein. The wild-type protein was found throughout the cytoplasm and neurites with a high degree of co-localization with the endoplasmic reticulum (ER) marker, protein disulfide isomerase. In contrast, the mutant protein accumulated in multiple, large inclusions in the cytoplasm around the nucleus. These inclusions were composed of membrane whorls, apparently derived from the ER. If disrupted processing of the mutant protein leads to its accumulation in multilayer membranous structures in vivo, these may interfere with membrane trafficking in neurons.

Impairment of sterol biosynthesis leads to phosphorus and calcium accumulation in Leishmania acidocalcisomes

The induction of the formation of inclusion vesicles in Leishmania amazonensis by the sterol biosynthesis inhibitors (SBI) ketoconazole and terbinafine has been reported previously. These compartments were recently identified as acidocalcisomes. By the use of electron spectroscopic imaging and energy loss spectroscopy, the presence of calcium, phosphorus and oxygen in the electron-dense inclusions located within the acidocalcisomes has been demonstrated. Endoplasmic reticulum cisternae formed membrane whorls which enclosed large portions of the cytoplasm and sometimes circumscribed acidocalcisomes. In addition, acid phosphatase activity, as well as the endocytic tracers horseradish peroxidase and gold-labelled transferrin and cystatin C were detected within these organelles in both SBI-treated and untreated parasites. These data suggest that impairment of sterol biosynthesis induces the biogenesis of acidocalcisomes and triggers an autophagic process that leads to intersection of the endosomal/lysosomal system with the acidocalcisomes.

The effect of aging on pars compacta of the substantia nigra in rhesus monkey.

This aim of this study was to re-evaluate the types of neurons present in pars compacta of the substantia nigra in the rhesus monkey, and then to determine the effects of aging on the morphology of both the neurons and neuroglial cells. The substantia nigra was therefore examined in Golgi impregnated material, in sections labeled with antibodies to tyrosine hydroxylase and to GABA, and in tissue embedded in plastic for light and electron microscopy. Three types of neurons were encountered: (1) large multipolar neurons with prominent Nissl bodies, (2) bipolar neurons that are medium sized and spindle-shaped, with Nissl bodies confined to the poles of the deeply indented nuclear envelopes, and (3) small multipolar cells with sparse Nissl substance. The large and medium neurons are believed to be dopaminergic and the small neurons GABA-ergic. With age, all of the neurons accumulate lipofuscin, especially the small multipolar neurons. In addition, Marinesco bodies appear within the nuclei of the large multipolar neurons. The dendrites are most severely affected by age; many of them lose organelles and their cytoplasm can become filled with vacuoles, membranous whorls, and dense bodies. In old monkeys many of the astrocytes have inclusions and in the neuropil there is a striking increase in the number of astrocytic processes. Also large spheroids appear. These appear to be derived from astrocytes and they have a core of dense granular material surrounded by a paler peripheral zone of cytoplasm from which processes can extend. Most of the oligodendrocytes have dense inclusions in their cytoplasm and many of the myelin sheaths break down. Microglial cells can become enormously swollen by phagocytosed material. Although both neurons and neuroglial cells are affected by age, no entities that could be construed to be dying neurons were encountered.

Ultrastructure of porcine circovirus in persistently infected PK-15 cells.

The ultrastructure of porcine circovirus was examined in persistently infected porcine kidney (PK)-15 cells. Virus-infected PK-15 cells had large numbers of intracytoplasmic inclusions, and a few cells had intranuclear inclusions. Intracytoplasmic inclusions were dispersed throughout the cytoplasm but were most numerous in the perinuclear cytoplasm. Inclusion were of various sizes, round to oval, and electron dense and were of two general types. Inclusions of the first type were small (0.1-0.5 microm diameter), not surrounded by trilaminar membranes, and granular with indistinct margins that blended with surrounding cytoplasm. Some contained 12+/-2-nm-diameter icosahedral virions in loose aggregates or rarely forming paracrystalline arrays. Small inclusions could be sites of viral assembly or maturation. Intracytoplasmic inclusions of the second type were larger (0.5-5.0 microm diameter) and more numerous and had abrupt margins surrounded by trilaminar membranes. They were more electron dense than small inclusions and were heterogeneous, containing various proportions of aggregated virions, electron-dense crystalline lamellae of 5 nm periodicity, and/or whorls of myelinoid membranes. Virions usually formed paracrystalline arrays and occasionally were loosely aggregated. Larger inclusions were typical of autophagolysosomes. Intranuclear inclusions were not membrane bound and were often associated with reticulated nucleoli or aggregates of heterochromatin. Some inclusions were irregularly shaped aggregates of indistinct, circular 10-12-nm-diameter viruslike particles. Others were 0.1-1.0 microm in diameter, round or ring shaped, dense, and finely granular, with sharply demarcated margins.

LIVER MITOCHONDRIA ALTERATIONS IN CHLOROFORM-TREATED SPRAGUE-DAWLEY RATS

The formation of a chloroform adduct produced by the reaction of the oxidative chloroform metabolite phosgene with two molecules of phosphatidylethanolamine has been previously demonstrated in liver mitochondria of phenobarbital-pretreated Sprague-Dawley (SD) rats. The aim of our study was to assess the influence of chloroform adduct mitochondrial accumulation on the hepatic mitochondria morphology. Liver mitochondrial ultrastructural alterations were analyzed by electron microscopy in SD rats administered with increasing doses of chloroform. Variation in the morphology of mitochondria, consisting of an increase of intertwined organelles, only rarely seen in control specimens, was observed at the lowest chloroform dose (180 mg/kg). At higher doses, mitochondrial damage progressed with swelling of the organelles and formation of megamitochondria. These megamitochondria were characterized by a dilution of the matrix, and often membranous whorls were found inside the matrix. The two distinct forms of cell death, necrosis and apoptosis, were first observed at 300 mg/kg of chloroform. Our results suggest that the formation and the accumulation of a chloroform-modified phosphatidylethanolamine in mitochondria induce ultrastructural modifications of these organelles. In conclusion, mitochondria are involved in chloroform-induced hepatotoxicity.

Ultrastructure of culture forms of the eel trypanosome, Trypanosoma granulosum Laveran & Mesnil, 1902, exposed to polyamine biosynthesis inhibitors

AbstractPolyamines are important in the growth, division and differentiation of many cell types, including trypanosomes. The ultrastructure of culture forms of Trypanosoma granulosum was examined following growth in a modified semidefined medium (controls) and in the same medium with added polyamine biosynthesis inhibitors (DFMO, MGBG and Berenil). Untreated trypanosomes had ultrastructural features in common with other cultured flagellates. Those treated with DFMO were generally more rounded in contour. Cytoplasmic vacuoles and swollen mitochondrial membranes suggested osmotic imbalance, perhaps resulting from compromised membrane integrity. An anticlockwise arm dividing subtubule B of the peripheral doublets of the flagellum was noted in some trypanosomes treated with 20 mM DFMO. Vacuolation was also induced by MGBG and flagellar division occurred more frequently at 0.2 mM than in controls. With 1 mM MGBG, mitochondria were difficult to discern and kinetoplasts were disrupted. With 0.2 mM Berenil, vacuoles, swollen mitochondria, membranous whorls, additional microtubules underlying subpellicular tubules and disaggregated kinetoplasts were noted. With 1 mM Berenil, much of the cell structure was destroyed, although the pellicle, flagellum and paraxial rod remained intact. The present study illustrates the importance of hnctional polyamine synthetic pathways for the integrity of membranes, mitochondria, kinetoplasts and possibly microtubules in cultured T. granulosum.

Processes of Blue Light–Induced Damage to Retinal Pigment Epithelial Cells Lacking Phagosomes

To experimentally clarify the processes of the changes induced by blue light directly on the retinal pigment epithelium (RPE) before the formation of phagosomes or the accumulation of lipofuscin. Methods: We developed a new experimental method in which primary cultured cells of very young pigmented rats were exposed to several intensities and durations of blue light (wavelength = 440 ± 10 nm). Results: At 1.0 mW/cm 2, the damage was limited to mitochondria. At 2.0 mW/cm 2, the cytoplasm exhibited large whorls of membrane or whorled inclusions, which were consistent with autophagic vacuoles. At 4.0 mW/cm 2, the RPE cells showed lysis of the cytoplasm and a nucleus that was consistent with necrosis. Conclusions: Our results suggested that damage induced by blue light to cultured RPE cells may originate in the mitochondria and end in necrosis. The type of cell death induced in the RPE by blue light seems to be determined mainly by the intensity of the light, but is also related to the duration of exposure.

Vacuolation in renal tubular epithelium of Cd-1 mice: an incidental finding.

Prominent cytoplasmic vacuoles were observed in renal tubular epithelial cells of the outer medulla in several kidneys from test article-dosed mice (Crl:CD-1 (ICR)BR VAF/PLUS) during routine light microscopic (LM) examination. Because the vacuolar change was detected infrequently and was not found in any control mice from that study, it was not clear whether the vacuolation represented a drug-induced change. To address this question, kidney sections from mice from multiple unrelated studies were examined by LM for similar vacuolar changes. Vacuolation was seen by LM in 2.3% of the control and 2.8% of the test article-dosed mice. Transmission electron microscopy (TEM) was also performed on kidneys with prominent light microscopic vacuoles in 5 control mice and 2 test article-dosed mice to further characterize the vacuoles. Ultrastructurally, the vacuoles contained fibrillar and finely stipled granular material or membranous whorls. Kidneys from control mice lacking light microscopic evidence of vacuolation had smaller vacuoles containing similar material when examined by TEM. Because vacuoles were present in both control mice and test article-dosed mice, it was concluded that the vacuoles were incidental and unrelated to compound administration. These studies also demonstrated that vacuoles can be expected to be observed by LM examination in 2-3% of Crl:CD-1 (ICR)BR VAF/PLUS, mice.

Observation of ultrastructural changes in cultured retinal pigment epithelium following exposure to blue light.

The retina can be damaged by light even when levels of energy are well below the threshold for thermal damage, and the experimental damage of the retinal pigment epithelium (RPE) may be induced more easily by blue light than by longer wavelengths of visible light. The present study demonstrates the ultrastructural damage produced by exposure to blue light in cultured RPE. Long-Evans rats were enucleated 8-10 days after birth for primary culture. One week after seeding, the monolayer culture of RPE cells was exposed to a cool blue light (wavelength = 440 +/- 10 nm) for 36 h (12 h/day, 3 days) at 2.0 mW/cm2. Transmission electron microscopy was used to compare the exposed RPE with the control. The entire experiment was repeated 3 times independently. The cytoplasm of the exposed RPE exhibited degenerative changes, such as large whorls of membrane, lamellar whorls and whorled inclusions. The RPE cells can be damaged directly by blue light after excluding the possible influence of phagosomes. This primary culture of RPE can also serve as an in vitro model for the study of light damage to the RPE.

The Nematode Degenerin UNC-105 Forms Ion Channels that Are Activated by Degeneration- or Hypercontraction-Causing Mutations

Nematode degenerins have been implicated in touch sensitivity and other forms of mechanosensation. Certain mutations in several degenerin genes cause the swelling, vacuolation, and death of neurons, and other mutations in the muscle degenerin gene unc-105 cause hypercontraction. Here, we confirm that unc-105 encodes an ion channel and show that it is constitutively active when mutated. These mutations disrupt different regions of the channel and have different effects on its gating. The UNC-105 channels are permeable to small monovalent cations but show voltage-dependent block by Ca2+ and Mg2+. Amiloride also produces voltage-dependent block, consistent with a single binding site 65% into the electric field. Mammalian cells expressing the mutant channels accumulate membranous whorls and multicompartment vacuoles, hallmarks of degenerin-induced cell death across species.

Ultrastructure of the binary parotid glands in the free-tailed bat, Tadarida thersites. II. Accessory parotid gland.

Many bat species have an extra set of major salivary glands. In some species, the accessory glands are quite similar to the principal one, but in others they may be radically different. Accessory glands usually are associated with the submandibular gland, but the free-tailed bat, Tadarida thersites, also has an accessory parotid gland. In the present study, we compared the accessory parotid gland with its principal counterpart. Salivary glands were removed from two specimens of free-tailed bats, one of each sex, that had been live-trapped in western Kenya and immersion-fixed in a specially formulated mixture designed for field fixation. Once back in the laboratory, the tissue was further prepared for electron microscopy by conventional means. The secretory endpieces consist of serous tubules composed of typical serous cells that contain numerous dense granules. In contrast, the intralobular duct system shows a radical departure from normal. These ducts are enormous in caliber, their lumina measuring greater than 100 microm in diameter. They appear to arise by amalgamation of the homologues of intercalated and striated ducts into macroducts. The walls of the macroducts consist of intermingled patches of simple cuboidal and simple columnar epithelia that occasionally include a tuft cell and are underlaid by an almost continuous layer of myoepithelium. A few cells have some modified basal striations, but most cells display a cytological organization that differs radically from either of their two putative ancestral cell types. Both tall and short epithelial macroduct cells have a paranuclear collection of ovate mitochondria and aggregates of what presumably are peroxisomes. Macroduct cells in both the female and male are pervaded by a system of tubular smooth endoplasmic reticulum (SER). In the female, the SER gives rise to membranous whorls that consist of numerous plies. As the macroducts approach their termini, a single row of small dense secretory granules appears just beneath their luminal surface. At the lobular periphery, the ducts taper down to become excretory ducts of normal dimensions. An accessory parotid gland occurs in T. thersites, but apparently is absent in the related species, T. brasiliensis. The ultrastructural data are consistent with a possible steroidogenic function, although other features of the gland might relate to the elaboration of a secretory product associated with feeding on chitinous beetles. The macroducts conceivably function as reservoirs of preformed saliva.

Further evidence of species variation in mechanisms of epithelial cell loss in mammalian small intestine: ultrastructural studies on the reindeer (Rangifer tarandus) and seal (Phoca groenlandica).

Ultrastructural studies were conducted on mechanisms of epithelial cell loss in the small intestine of seal and reindeer. Mechanisms maintaining epithelial integrity were distinguished from those that did not and the non-epithelial cell types involved were identified. Three types of cell extrusion were noted. In two, tight junctional integrity was preserved and anucleate apical cell fragments (rather than complete cells) were lost into the lumen. In reindeer (type 1), this involved creating large intercellular spaces extending from the preserved apical cap to the lamina propria and containing enterocyte debris probably phagocytosed by subepithelial macrophages. A variant of this process (type 2) involved the gradual shrinkage of individual cells, which became more electron-dense, and the in situ degeneration of their nucleated subapical portions. Degenerated cell fragments and membrane whorls were confined to narrow intercellular spaces between approximating adjacent healthy enterocytes. The mechanism of removal of these fragments was unclear. In both cases, the proximity of intraepithelial lymphocytes suggested that they were involved in cell targetting and killing. Evidence of apoptotic nuclei was not found but nucleated cell fragments could have been washed out of the lumen during tissue preparation. Type 2 cell loss was seen in both species, as was another mechanism (type 3) reminiscent of necrosis. In contrast to other mechanisms, this was accompanied by breaks in epithelial continuity following gradual loss of cell electron density and total or subtotal degradation of organelles and membranes. In seal, this terminated in the loss of an abnormal cell apex and exposure of the contents of the cell remnant to the lumen. In reindeer, all the cell remnants may have been extruded before total membrane degeneration but, in both species, the otherwise tight epithelial barrier was clearly breached. Again, intraepithelial lymphocytes were associated with sites of necrosis. These findings provide evidence for further species differences in mechanisms of epithelial cell extrusion and suggest that necrotic cell loss may be more common than previously admitted.

Different concentrations of ascorbyl-2-monophosphate-magnesium as dietary sources of vitamin C for seabass, Lates calcarifer

In this study, the effects of different concentrations of ascorbyl-2-monophosphate-magnesium (APM; 0, 30, 60, or 100 mg kg −1 diet) were tested in seabass for 6 weeks. Fish fed the diet without vitamin C supplementation showed various clinical signs such as fin erosion, erratic swimming behavior and spinal deformities. Several pathological changes were found in the gills and kidneys of fish fed a diet deficient in APM. The most remarkable feature in the gills was deformation of the outer epithelial cell layer, separation of the epithelial tissues of the secondary lamellae, and hyperplasia of the respiratory epithelium. An accumulation of red blood cells in the space between the outer and inner layer of epithelial cells of secondary lamellae was also observed. Changes in the kidneys were found in fish fed the APM-free diet; these changes included swelling, deformation, and fusion of mitochondria, as well as membrane whorls of endoplasmic reticulum. Seabass fed APM-containing diets did not show any of these abnormalities in the gills or proximal tubule cells of the kidney.

Neuropathology of Degenerative Cell Death inCaenorhabditis elegans

In Caenorhabditis elegans necrosis-like neuronal death is induced by gain-of-function (gf) mutations in two genes, mec-4 and deg-1, that encode proteins similar to subunits of the vertebrate amiloride-sensitive epithelial Na+ channel. We have determined the progress of cellular pathology in dying neurons via light and electron microscopy. The first detectable abnormality is an infolding of the plasma membrane and the production of small electron-dense whorls. Later, cytoplasmic vacuoles and larger membranous whorls form, and the cell swells. More slowly, chromatin aggregates and the nucleus invaginates. Mitochondria and Golgi are not dramatically affected until the final stages of cell death when organelles, and sometimes the cells themselves, lyse. Certain cells, including some muscle cells in deg-1 animals, express the abnormal gene products and display a few membrane abnormalities but do not die. These cells either express the mutant genes at lower levels, lack other proteins needed to form inappropriately functioning channels, or are better able to compensate for the toxic effects of the channels. Overall, the ultrastructural changes in these deaths suggest that enhanced membrane cycling precedes vacuolation and cell swelling. The pathology of mec-4(gf) and deg-1(gf) cells shares features with that of genetic disorders with alterations in channel subunits, such as hypokalemic periodic paralysis in humans and the weaver mouse, and with degenerative conditions, e.g., acute excitotoxic death. The initial pathology in all of these conditions may reflect attempts by affected cells to compensate for abnormal membrane proteins or functions.

Age-Dependent Neuronal and Synaptic Degeneration in Mice Transgenic for the C Terminus of the Amyloid Precursor Protein

The molecular basis for the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature in Alzheimer's disease (AD) is incompletely understood. We have proposed that one molecule common to these abnormal processes is a fragment of the Alzheimer amyloid precursor protein (APP) comprising the C-terminal 100 amino acids of this molecule (APP-C100). We tested this hypothesis by creating transgenic mice expressing APP-C100 in the brain. We report here that aging (18-28 month) APP-C100 transgenic mice exhibit profound degeneration of neurons and synapses in Ammon's horn and the dentate gyrus of the hippocampal formation. Of the 106 transgenic mice between 8 and 28 months of age that were examined, all of those older than 18 months displayed severe hippocampal degeneration. The numerous degenerating axonal profiles contained increased numbers of neurofilaments, whorls of membrane, and accumulations of debris resembling secondary lysosomes near the cell body. The dendrites of degenerating granule and pyramidal cells contained disorganized, wavy microtubules. Cerebral blood vessels had thickened refractile basal laminae, and microglia laden with debris lay adjacent to larger venous vessels. Mice transgenic for Flag-APP-C100 (in which the hydrophilic Flag tag was fused to the N terminus of APP-C100) showed a similar degree of neurodegeneration in the hippocampal formation as early as 12 months of age. The 45 control mice displayed only occasional necrotic cells and no extensive cell degeneration in the same brain regions. These findings show that APP-C100 is capable of causing some of the neuropathological features of AD.

Cytopathology of Bay of Piran Shrimp Virus (BPSV), a New Crustacean Virus from the Mediterranean Sea

A new picorna-like or parvo-like virus was found in the hepatopancreas of the shrimpPalaemon elegansfrom the Bay of Piran (Mediterranean Sea). Bay of Piran shrimp virus (BPSV) infects preferably the R-cells of the hepatopancreas and to a lesser extent the F-cells. In R-cells it is abundant in the cytoplasm and within membrane whorls. In lesser numbers it can also be present in cell organelles like the endoplasmic reticulum, autophagosomes, or mitochondria. Less contrasting virus-like particles are observed in the nucleoplasm, suggesting a nuclear replication of the virus and completion of assembly in the cytoplasm. In F-cells the virus is located mostly in cisternae of the Golgi bodies. Occasionally, there are also virus clusters in the cytoplasm. B-cells are only rarely infected. The virus seems to invade the host cells from the lumen of the hepatopancreatic tubules through the microvillous border. Mature viruses are released again into the hepatopancreatic lumen by discharge of lytic epithelial cells. Individual viruses can also penetrate through the basal cell membrane into the hemal space. Occasionally, fixed phagocytes are attached to the basal lamina of heavily infected hepatopancreas cells, indicating an activation of the immune defense system by the virus.

Ultrastructural consequences of radical damage before and after differentiation of neuroblastoma B-104 cells

There is abundant evidence that the pathophysiology leading to neuronal death during post-ischemic brain reperfusion involves radical-mediated damage. Although the ultrastructural alterations accompanying brain ischemia and reperfusion are well characterized, little is known about the ultrastructural alterations that are specific to radical damage. This study examines in differentiated and undifferentiated neuroblastoma B-104 cells the viability (by dye exclusion) and ultrastructural consequences of radical damage initiated by 50 microM cumene hydroperoxide (CumOOH). Differentiation was most notably associated with formation of neurites and an extensive cytoskeletal feltwork. CumOOH-induced cell death was increased after differentiation and was blocked by the iron chelator DETAPAC. The ultrastructural characteristics of radical damage here included: (1) plasmalemmal holes that appear to undergo "patching" by well-organized membrane whorls, (2) accumulation of numerous free ribosomes, (3) markedly increased vesicular trafficking about the Golgi accompanied by Golgi transformation from cisternal organization to clusters of vacuoles with numerous fusing vesicles, (4) development of large multi-layered vacuoles that include damage membranes and organelles and appear to undergo extrusion from the cell, and (5) a general loss of cytoplasmic volume. These ultrastructural alterations developed more rapidly and were consistently more advanced in differentiated cells throughout the 6-h time course. In differentiated cells radical damage also induced the disorganization and subsequent loss of the extensive feltwork of cytoskeletal elements. There was little damage to the membranes of the nuclear envelope and mitochondria. Our observations in this system are strikingly similar to ultrastructural alterations in Golgi and ribosomal organization seen in vulnerable neurons during post-ischemic brain reperfusion and suggest that these alterations during reperfusion reflect the consequence of radical-mediated damage.

Clearance of bacteria injected into the hemolymph of the ridgeback prawn, Sicyonia ingentis (Crustacea: Decapoda): Role of hematopoietic tissue.

In an earlier investigation, radiolabelled bacteria injected into the hemolymph of the shrimp Sicyonia ingentis were cleared rapidly from circulation. Most of the bacteria were localized in the gills, followed by other organs including the heart, abdominal musculature, and hematopoietic nodules (HPN). We determined the organ-specific effectiveness of bacterial clearance and found the HPN to be the most effective on a per gram basis. Light and electron microscopy were used to determine the mechanism of clearance in the HPN. The HPN are readily permeable to individual bacteria, which then are recognized and phagocytosed exclusively by small granule hemocytes. Within the first hour, the bacteria are degraded, leaving only whorls of membrane in the phagocytic vacuoles. Subsequently, the hemocytes that ingested bacteria lyse, as has been observed in vitro. These observations support earlier suggestions that hemocytes in the HPN are functionally mature and held in reserve until they are released into circulation. © 1996 Wiley-Liss, Inc.