Membranous Whorls Research Articles

The C Terminus of Peripherin/rds Participates in Rod Outer Segment Targeting and Alignment of Disk Incisures

Protein targeting is essential for domain specialization in polarized cells. In photoreceptors, three distinct membrane domains exist in the outer segment: plasma membrane, disk lamella, and disk rim. Peripherin/retinal degeneration slow (rds) and rom-1 are photoreceptor-specific members of the transmembrane 4 superfamily of transmembrane proteins, which participate in disk morphogenesis and localize to rod outer segment (ROS) disk rims. We examined the role of their C termini in targeting by generating transgenic Xenopus laevis expressing green fluorescent protein (GFP) fusion proteins. A GFP fusion containing residues 317-336 of peripherin/rds localized uniformly to disk membranes. A longer fusion (residues 307-346) also localized to the ROS but exhibited higher affinity for disk rims than disk lamella. In contrast, the rom-1 C terminus did not promote ROS localization. The GFP-peripherin/rds fusion proteins did not immunoprecipitate with peripherin/rds or rom-1, suggesting this region does not form intermolecular interactions and is not involved in subunit assembly. Presence of GFP-peripherin/rds fusions correlated with disrupted incisures, disordered ROS tips, and membrane whorls. These abnormalities may reflect competition of the fusion proteins for other proteins that interact with peripherin/rds. This work describes novel roles for the C terminus of peripherin/rds in targeting and maintaining ROS structure and its potential involvement in inherited retinal degenerations.

Perinuclear biogenesis of mutant torsin-a inclusions in cultured cells infected with tetracycline-regulated herpes simplex virus type 1 amplicon vectors

TorsinA is a novel protein identified in the search for mutations underlying the human neurologic movement disorder, early onset torsion dystonia. Relatively little is understood about the normal function of torsinA or the physiological effects of the codon deletion associated with most cases of disease. Overexpression of wild-type torsinA in cultured cells by DNA transfection results in a reticular distribution of immunoreactive protein that co-localizes with endoplasmic reticulum resident chaperones, while the dystonia-related mutant form accumulates within concentric membrane whorls and nuclear-associated membrane stacks. In this study we examined the biogenesis of mutant torsinA-positive membrane inclusions using tetracycline-regulated herpes simplex virus amplicon vectors. At low expression levels, mutant torsinA was localized predominantly around the nucleus, while at high levels it was also concentrated within cytosolic spheroid inclusions. In contrast, the distribution of wild-type torsinA did not vary, appearing diffuse and reticular at all expression levels. These observations are consistent with descriptions of inducible membrane synthesis in other systems in which cytosolic membrane whorls are derived from multilayered membrane stacks that first form around the nuclear envelope. These results also suggest that formation of mutant torsinA-positive inclusions occurs at high expression levels in culture, whereas the perinuclear accumulation of the mutant protein is present even at low expression levels that are more likely to resemble those of the endogenous protein. These nuclear-associated membrane structures enriched in mutant torsinA may therefore be of greater relevance to understanding how the dystonia-related mutation compromises cellular physiology.

Proliferative growth of SARS coronavirus in Vero E6 cells.

An isolate of SARS coronavirus (strain 2003VA2774) was obtained from a patient and used to infect Vero E6 cells. The replication cycle of the virus was followed from 1 to 30 h post-infection (p.i.). It was surprising to observe the swift growth of this human virus in Vero cells. Within the first hour of infection, the most obvious ultrastructural change was the proliferation of the Golgi complexes and related vesicles accompanied by swelling of some of the trans-Golgi sacs. Extracellular virus particles were present by 5 h p.i. in about 5 % of the cells and this increased dramatically to about 30 % of the cell population within an hour (6 h p.i.). Swollen Golgi sacs contained virus nucleocapsids at different stages of maturation. These virus precursors were also in large vacuoles and in close association with membrane whorls. The membrane whorls could be the replication complexes, since they appeared rather early in the replication cycle. As infection progressed from 12 to 21 h p.i., the cytoplasm of the infected cells was filled with numerous large, smooth-membraned vacuoles containing a mixture of mature virus and spherical cores. Several of these vacuoles were close to the cell periphery, ready to export out the mature progeny virus particles via exocytosis. By 24 to 30 h p.i., crystalline arrays of the extracellular virus particles were seen commonly at the cell surface.

Early events of SARS coronavirus infection in vero cells.

An isolate from a patient in the recent severe acute respiratory syndrome (SARS) outbreak in Singapore was used to infect Vero E6 cells. This study concentrated on the first 30 min of infection. It was discovered that the SARS coronavirus attached, entered, and uncoated the nucleocapsids, all within a 30‐min period. At 5 min after infection, several virus particles lined the Vero cell plasma membrane. Virus particles were at various stages of fusion at the cell surface, since entry was not a synchronised process. After entry (10 and 15 min), spherical core particles moved into the cytoplasm within large vacuoles. Quite surprising at such early stages of infection (20 min), a virus‐induced change in the infected cells was evident. The induction of myelin‐like membrane whorls was obvious within the same vacuoles as the core particles. The significance of this virus‐induced change is unknown at this stage. By 25–30 min postinfection (p.i.), the spherical core particles appeared to be disassociating and, in their place, doughnut‐shaped electron‐dense structures were observed. These could be the virus genomes together with the helical nucleocapsids. They were no longer in large vacuoles but packaged into smaller vacuoles in the cytoplasm, and occasionally in small groups. J. Med. Virol. 71:323–331, 2003. © 2003 Wiley‐Liss, Inc.

Cellular responses to increasing Cd concentrations in the freshwater crab, Potamonautes warreni, harbouring microbial gill infestations.

We investigated the uptake, transport, storage and defence mechanisms in the freshwater crab, Potamonautes warreni, harbouring microbial gill infestations and exposed to increasing chronic (0.2, 0.5, 1.0 mg l(-1)) and acute (2.0 mg l(-1)) cadmium (Cd) concentrations under controlled laboratory conditions over a period of 21 days. Transmission electron microscopy and X-ray microanalysis revealed that the microbial gill fauna was eliminated on exposure to 0.2 mg Cd(2+) l(-1) and that Cd became increasingly adsorbed and incorporated into lamellar crystal deposits and permeated the cuticle of the gills of P. warreni. Degeneration of the apical membrane infoldings and vacuolation of epithelial cells occurred concurrently with pinocytosis, endocytosis and pronounced phagocytotic activity in the epithelia and haemal canal of the gills. Elevated Cd exposures (0.5 or 1.0 mg l(-1)) resulted in the swelling and dissociation of mitochondrial outer membranes together with an increase in transport of Cu, Cl and S by haemocytes in the haemal canal to epithelial tissues depleted in these elements. Cd also accumulated in tightly coiled concentric membrane whorls in the haemal canal, whereas the highest concentrations of Cd were found within aggregates of lysosome-like bodies in cuticulin-secreting cells of the gill stem. Chronic exposure to Cd induced increased fatigue and mild uncoordinated motor activity. In contrast, at an acute exposure of 2.0 mg l(-1) over 48 h, P. warreni showed a time-specific rapid loss of motor function, although only mild cellular lesions occurred in the gill tissues. The significance of cellular changes in the gill epithelia and altered motor activity of P. warreni with increased waterborne Cd are discussed as potential biomarker responses in monitoring aquatic pollution.

PIKfyve Controls Fluid Phase Endocytosis but Not Recycling/Degradation of Endocytosed Receptors or Sorting of Procathepsin D by Regulating Multivesicular Body Morphogenesis

The mammalian phosphatidylinositol (PtdIns) 5-P/PtdIns 3,5-P2-producing kinase PIKfyve has been implicated in maintaining endomembrane homeostasis in mammalian cells. To address the role of PIKfyve in trafficking processes, we examined the functioning of the biosynthetic, endocytic, and recycling pathways in stable human embryonic kidney 293 cell lines inducibly expressing the wild-type or kinase-defective dominant-negative form. PIKfyveWT or PIKfyveK1831E expression did not affect the processing and lysosomal targeting of newly synthesized procathepsin D. Likewise the rates of transferrin uptake/recycling or epidermal growth factor receptor degradation were not altered upon expression of either protein. In contrast, PIKfyveK1831E but not PIKfyveWT expression markedly impaired the late uptake of fluid phase marker horseradish peroxidase. Inspection of the organelle morphology by confocal microscopy with specific markers in COS cells transiently expressing PIKfyveK1831E showed the Golgi apparatus, end lysosomes, and the recycling compartment indistinguishable from nontransfected cells, despite the dramatic PIKfyveK1831E-induced endomembrane vacuolation. In contrast, we observed a striking effect on the late endocytic compartment, marked by disruption of the dextran-labeled perinuclear endosomal compartment and formation of dispersed enlarged vesicles. Electron microscopy identified the cytoplasmic vacuoles in the PIKfyveK1831E-expressing human embryonic kidney 293 cells as enlarged multivesicular body-like structures with substantially lower number of internal vesicles and membrane whorls. Together, these data indicate that PIKfyve selectively regulates the sorting and traffic of peripheral endosomes containing lysosomaly directed fluid phase cargo through controlling the morphogenesis and function of multivesicular bodies.

Evidence for trafficking of PfEMP1 to the surface of P. falciparum-infected erythrocytes via a complex membrane network

The human malarial parasite Plasmodium falciparum exports virulence determinants, such as the P. falciparum erythrocyte membrane protein 1 (PfEMP1), beyond its own periplasmatic boundaries to the surface of its host erythrocyte. This is remarkable given that erythrocytes lack a secretory pathway. Here we present evidence for a continuous membrane network of parasite origin in the erythrocyte cytoplasm. Co-localizations with antibodies against PfEMP1, PfExp-1, Pf332 and PfSbpl at the light and electron microscopical level indicate that this membrane network is composed of structures that have been previously described as tubovesicular membrane network (TVM), Maurer's clefts and membrane whorls. This membrane network could also be visualized in vivo by vital staining of infected erythrocytes with the fluorescent dye LysoSensor Green DND-153. At sites where the membrane network abuts the erythrocyte plasma membrane we observed small vesicles of 15-25 nm in size, which seem to bud from and/or fuse with the membrane network and the erythrocyte plasma membrane, respectively. On the basis of our data we hypothesize that this membrane network of parasite origin represents a novel secretory organelle that is involved in the trafficking of PfEMP1 across the erythrocyte cytoplasm.

Ultrastructural changes in Chlamydomonas acidophila (Chlorophyta) induced by heavy metals and polyphosphate metabolism

Ultrastructural changes induced by heavy metals (cadmium, zinc, and copper) and polyphosphate metabolism were studied in Chlamydomonas acidophila. Transmission electron microscopy indicated that cadmium led to the most drastic morphometric changes. An increase in number and volume of starch grains and vacuoles as well as the presence of electron dense deposits in vacuole and membrane whorls were observed. Energy-dispersive X-ray analysis revealed that vacuolar deposits inside cells treated with cadmium contained phosphate and cadmium. These ultrastructural changes were accompanied by a change in the intracellular polyphosphate level, as shown by in vivo 31P-nuclear magnetic resonance. It was also observed that cadmium treatment caused polyphosphate degradation and increased vacuolar short-chains and orthophosphates.

An RCS-like retinal dystrophy phenotype in mer knockout mice.

To determine whether mice that are homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (mer(kd)) manifest a retinal dystrophy phenotype similar to RCS rats, which carry a mutation in the orthologous gene MERTK: Eyes of mer(kd) and C57BL/6 wild-type (WT) mice were examined by light and electron microscopy, whole-eye rhodopsin measurement, and Ganzfeld electroretinography (ERG). The mer(kd) mice showed rapid, progressive degeneration of the photoreceptors (PRs). Features of the phenotype common to mer(kd) mice and RCS rats included the absence or near absence of phagosomes in the retinal pigment epithelium (RPE) at the peak of outer segment (OS) disc shedding, accumulation of debris and whorls of membranes at the RPE-OS interface, transient supernormal rhodopsin content and OS lengths, the presence of OS vacuoles beginning at early ages, and a relatively slow removal of pyknotic PR nuclei. Most PRs were missing, and OS debris was removed by approximately postnatal day (P)45. Scotopic ERG responses were lower than age-matched WT responses and declined with PR loss. Photopic responses were preserved better than scotopic responses, corresponding with preferential cone preservation as judged histologically. ERG amplitudes were usually unmeasurable beyond P40, although a small-amplitude scotopic threshold response (STR) could still be elicited at P253 in some mice when only scattered PR nuclei remained. Ablation of Mer function in mer(kd) mice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK.

Inherited retinal dystrophy in Mer knockout mice.

Abnormalities of the retinal pigment epithelial (RPE) cells are seen in several inherited degenerations such as Best disease (Petrukhin et al., 1998; Marmorstein et al., 2000), Stargardt disease (Weng et al., 1999), Sorsby’s fundus dystrophy (Steinmetz et al., 1992; Jacobson et al., 1995), and childhood-onset severe retinal dystrophy (Gu et al., 1997) and Leber congenital amaurosis due to RPE65 mutations (Marlhens et al., 1997; Morimura et al., 1998; Lotery et al., 2000; Thompson et al., 2000). RCS rats have been studied extensively as a model of photoreceptor (PR)-RPE cell interactions (Mullen and LaVail, 1976; LaVail, 2001) and retinal degeneration (Strauss et al., 1998; LaVail, 2001). Retinal degeneration in RCS results from failure of the RPE to phagocytize shed rod outer segments (OS) (Herron et al., 1969; Bok and Hall, 1971). The unphagocytized OS membranes form membranous whorls at the RPE surface, and the rod OS grow abnormally long (Dowling and Sidman, 1962; LaVail and Battelle, 1975). Eventually, the OS layer degenerates into a debris zone with subsequent PR cell death (Dowling and Sidman, 1962).KeywordsRetinal DegenerationRetinal DystrophyStargardt DiseaseVitelliform Macular DystrophyScotopic Threshold ResponseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Histidine decarboxylase deficiency in gene knockout mice elevates male sex steroid production.

Histamine is synthesized in cells by histidine decarboxylase (HDC). HDC-deficient knockout (KO) mice lack functional HDC and histamine in the tissues. In the present study we used this in vivo model for studying the role of HDC deficiency in the regulation of male steroid hormone metabolism. In agreement with earlier studies showing the lack of effects of central histamine on the basal secretion of gonadotrope hormones, we found no difference with in situ hybridization in the expression of GnRH in the hypothalamus of wild type and KO mice. The tissue concentrations of testosterone and several androgenic steroids were significantly elevated in the testes but not in the adrenal glands of HDC-KO mice. In contrast, serum estradiol levels failed to show a significant difference between the two groups. The weight of the testes was significantly smaller in both 7-day-old and adult KO mice. The ultrastructure of the adult testis indicated elevated steroid synthesis with more tightly coiled membranous whorls in Leydig cells. The present results suggest that changes in reproductive functions and sex steroid secretion in male HDC-KO mice are not due to altered hypothalamic GnRH expression but are probably related to definite modifications during fetal development of KO mice reinforced later by the lack of the effect of peripheral histamine. This may provide in vivo evidence that peripheral histamine is an important regulatory factor of male gonadal development during embryogenesis and of sex steroid metabolism later in adulthood.

Striatal postsynaptic ultrastructural alterations following methylenedioxymethamphetamine administration.

Amphetamine derivatives, such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA), act as monoaminergic neurotoxins in the central nervous system. Although there are slight differences in their mechanism of action, these compounds share a final common pathway, which involves dopamine release and oxidative stress. Apart from striatal toxicity involving monoamine axons, no previous report evidenced any alteration at the striatal level concerning postsynaptic sites. Given the potential toxicity for extracellular dopamine at the striatal level, and the hypothesis for neurotoxic effects of dopamine on striatal medium-sized neurons in Huntington's disease, we evaluated at an ultrastructural level the effects of MDMA on intrinsic striatal neurons of the mouse. In this study, administering MDMA, we noted ultrastructural alterations of striatal postsynaptic GABAergic cells consisting of neuronal inclusions shaped as whorls of concentric membranes. These whorls stained for ubiquitin but not for synuclein and represent the first morphologic correlate of striatal postsynaptic effects induced by MDMA.

Ultrastructural pathology of Baltic salmon, Salmo salar L., yolk sac fry with the M74 syndrome

The ultrastructural pathology in liver, brain, skeletal and cardiac muscle of Baltic salmon yolk sac fry with the M74 syndrome is described. In the clinical stage of disease, the main pathological findings in the liver were a depletion of glycogen, condensation of nuclear chromatin, hydropic degeneration of mitochondria and a dilation of the bile canaliculi. In the terminal stage, additional findings were lipid accumulation and myelin whorls in the cytoplasm. The rough endoplasmic reticulum (RER) was degranulated and vesiculated and in some individuals, it formed concentric membranous whorls. Mitochondria showed several additional lesions, such as matrix densities, pleomorphism and cristae abnormalities. Skeletal myocytes were degenerated, and intracellular lipid accumulation was seen in the myocardium. In the brain, an increased frequency of cells exhibiting pyknosis or karryorhexis was recorded. The cytoplasm of these cells formed an amorphous mass of moderate density. The evaluation of brain and skeletal muscle was complicated by sporadic occurrence of pathological findings in the reference material, i.e. clinically healthy Baltic salmon yolk sac fry. As these yolk sac fry are suspected to have a subclinical thiamine deficiency, reference material in future studies should include salmon yolk sac fry from Atlantic populations or originating from reared broodstock.

Spinosad insecticide: subchronic and chronic toxicity and lack of carcinogenicity in Fischer 344 rats.

Spinosad is an insecticide derived from a naturally occurring bacterium via fermentation. The toxicity of spinosad was characterized in subchronic and chronic toxicity/oncogenicity studies conducted according to standard toxicology regulatory guidelines. Subchronic toxicity was evaluated in groups of 10 Fischer 344 rats/sex given feed containing 0, 0.05, 0.1, 0.2, or 0.4% spinosad (Study 1) or 0, 0.003, 0.006, 0.012, or 0.06% spinosad (Study 2) for 13 weeks. Lower body weights and increased mortality occurred in rats given 0.4% spinosad. Microscopic effects were observed in the adrenal glands, liver, lymphoid cells, reproductive tissues, kidney, thyroid, stomach, lung, and skeletal muscle of rats given > or = 0.05% spinosad, and consisted primarily of vacuolation of cells; however, degenerative, regenerative, and/or inflammatory changes were also noted in some tissues. Vacuolation within a number of tissues was ultrastructurally characterized by an increase in size and number of lysosomes that contained extensive membranous whorls consistent with phospholipidosis. The no observed effect level (NOEL) in the 13-week studies was 0.012% (24 mg/kg/day) spinosad. Chronic toxicity and oncogenicity were evaluated in groups of 60 Fischer 344 rats/sex given feed containing 0, 0.005, 0.02, 0.05, or 0.1% spinosad for up to 2 years. Rats given 0.1% spinosad for 1 year had microscopic effects similar to those observed in the subchronic studies. Vacuolation and inflammation of the thyroid gland also occurred in rats given 0.05% spinosad for 1 year. Excessive mortality occurred in rats from the oncogenicity study given 0.1% spinosad by 21 months, and surviving rats were euthanized because the maximum tolerated dose had been exceeded. Rats given 0.05% spinosad for 2 years had vacuolation and/or inflammation involving the thyroid, lymphoid tissue, and lung. Rats given 0.05% spinosad had similar numbers of neoplasms as control rats, indicating that spinosad was not carcinogenic at dose levels up to 0.05%. The NOEL at 2 years was 0.005% (2.4 mg/kg/day) spinosad.

Growth, structure, stomatal responses and secondary metabolites of birch seedlings ( Betula pendula) under elevated UV-B radiation in the field

Three-year-old birch (Betula pendula Roth.) seedlings were exposed, in the field, to supplemental levels of UV-B radiation. Control seedlings were exposed to ambient levels of UV radiation, using arrays of unenergized lamps. A control for UV-A radiation was also included in the experiment. Enhanced UV-B radiation had no significant effects on height growth, and shoot and root biomass of birch seedlings. Leaf expansion rate increased transiently in the middle of the growing period in enhanced UV-B- and UV-A-exposed plants; however, final leaf size and relative growth rate remained unaffected. Leaf thickness and spongy intercellular spaces were increased in UV-B-exposed seedlings along with increased density of glandular trichomes. At the ultrastructural level, enhanced UV-B increased the number of cytoplasmic lipid bodies, and abnormal membrane whorls were found. Both enhanced UV-B and UV-A radiation induced swelling of chloroplast thylakoids. Stomatal density and conductance were significantly increased by elevated UV-B radiation. UV-A radiation increased the length and width of stomata, whereas UV-B radiation had only a marginal effect on stomatal size. UV-A and enhanced UV-B radiation attenuated the appearance of necrotic spots in autumn, probably caused by the fungus Pyrenopeziza betulicola, suggesting a direct harmful effect of UV on pathogens or reduced susceptibility to pathogens in UV-exposed seedlings. Secondary metabolite analysis showed increases in (+)-catechin, quercetin, cinnamic acid derivative, apigenin and pentagalloylglucose in birch leaves under enhanced UV-B radiation. Negative correlations between apigenin, and particularly quercetin concentrations and lipid peroxidation levels indicated an antioxidant role of secondary metabolites in birch leaves exposed to UV-B radiation.

Lysosomes and sulfide-oxidizing bodies in the bacteriocytes of Lucina pectinata , a cytochemical and microanalysis approach

Lucina pectinata is a large tropical clam living deeply burrowed in the black, reducing mud of mangrove swamps. It is known to possess hemoglobin in the cytoplasmic areas of its bacteriocytes, which harbor sulfide-oxidizing bacteria. The bacteriocytes also possess lysosome-like microbodies containing either membrane whorls or electron-dense granules in which free heme compounds have been identified. The cytochemical detection of acid phosphatase and arylsulphatase through EDX (energy-dispersive X-ray) microanalysis strongly suggests that the bacteriocytes of L. pectinata contain, in fact, two different types of microbodies. Some of these (devoid of dense granules) possess a variable amount of lysosomal enzymes and occasionally a limited quantity of iron, which may result from a recycling process of hemoglobin. Their main function seems to be the digestion of a limited proportion of symbiotic bacteria. They represent genuine secondary lysosomes with a functionally acidic pH. The second type of microbodies is characterized by dense granules containing sulfur and iron hemes but no lysosomal enzymes. Their sulfide-oxidizing activity was substantiated by benzyl viologen assay, with Na2S as a substrate. These microbodies appear to be similar to the sulfide-oxidizing bodies (SOBs) described in the bacteriocytes of other bivalve species with symbiotic thioautotrophic bacteria; however, their sulfide-oxidizing activity appears to be non-enzymatic. They are discrete organelles, characterized by a functionally basic pH and pseudoperoxidasic activity, and have been termed SOBs. Therefore, the bacteriocytes of L. pectinata possess at the same time functional lysosomes and functional SOBs.

Histological diagnosis of mucopolysaccharidosis IIIA in a wire-haired dachshund

A four-year-old wire-haired dachshund developed progressive neurological signs of ataxia, intention tremor and finally dysuria. Two years later, histopathology showed that neurons throughout the brain and spinal cord were distended...

A lysosomal storage disease of Romney sheep that resembles human type 3 GM1 gangliosidosis.

During a large neuropathological survey of clinically normal sheep for evidence of scrapie, three adult Romney ewes from the same farm were found to have populations of distended neurons containing granular eosinophilic storage material. The affected neurons were confined to the striatum. The granules had the tinctorial properties of glycolipid and the ultrastructural appearance of spherical bodies containing concentric membranous whorls. The bodies resembled those of GM1 gangliosidosis. The restricted neuroanatomical distribution of changes together with the age of the sheep suggests a similarity to human type 3 GM1 gangliosidosis, which has not previously been reported in animals.

Necrotic Neurodegeneration in Caenorhabditis Elegans.

Necrosis plays a central role in neuronal injury associated with stroke and ischemia. Unlike apoptotic cell death, little is understood of molecular mechanisms of necrosis. The two distinct forms of cell death, necrosis and apoptosis, are easily distinguishable by morphological characteristics in the nematode Caenorhabditis elegans. At the level of light microscopy, necrotic cells appear as swollen vacuoles several times larger than normal cells; apoptotic cells become compacted and raised. Under the electron microscope the first visible step in necroticlike death is the involution of the plasma membrane, followed by the appearance of membrane whorls [1]. Later, the electron density of the cytoplasm is reduced, the cell swells and cytoplasmic vacuoles arise. The chromatin forms aggregates and becomes fragmented. Organelles swell and finally lyse. The dead cell is removed by phagocytosis [2]. The key characterized initiators and regulators of apoptotic cell death do not influence the initiation or progression of necrotic cell death [2]. A variety of different insults can initiate necrotic-like neurodegeneration in C. elegans and lead to similar morphological changes in the affected cells. Necrosis-inducing genes include dominant alleles of the mec-4 and deg-1 degenerins which encode ion-channel subunits with similarity to mammalian epithelial Na + -ion channels (ENaC) [3, 4, 5], a dominant allele of the deg-3 acetylcholine receptor α-subunit [6], and a transgenically expressed constitutively activated mutant of the G-protein subunit GαS [7]. That channel hyperactivating mutations induce necrosis is reminiscent of the initiation of excitotoxic cell death in humans. In our lab we study necrotic neurodegeneration with a focus on channel-hyperactivating mutations in the mec-4 gene as the death-initiating event. The MEC-4 protein is expressed in the six touch neurons in C. elegans, where it participates in the formation of mechanically gated ion channels and is involved in the signal transduction of mechanical stimuli. The dominant mec-4(d) allele encodes a hyperactive channel that leads to necrotic demise of the touch receptor neurons. We established a behavioral assay that allowed us to isolate suppressor mutations that block mec-4(d)-induced neurodegeneration. Ectopic expression of mec-4(d) in the ventral nerve cord leads to the appearance of a number of swelling and degenerating cells visible as vacuoles during the larval stage L1. As a consequence of this cell death, worms are severely paralyzed. We mutagenized 45.000 haploid genomes and isolated 24 worms with reconstituted locomotion. Such strains harbor candidate suppressors of mec-4(d)-induced neurodegeneration. Ten of the suppressors proved to be alleles of mec-6, which is a known suppressor of ectopically expressed mec-4(d). The other loci define several previously unknown genes involved in mec-4(d)-induced cell death.

Cytological alterations in isolated hepatocytes from common carp (Cyprinus carpio L.) exposed to microcystin-LR.

Microcystin-LR (MC-LR) is the most commonly encountered of the toxic cyclic peptide hepatotoxins occurring in China. It is a model compound for toxicological studies. In this study, the toxicity of MC-LR (50 and 500 micrograms/L) on isolated carp hepatocytes was determined and the ultrastructural alterations of cells induced by MC-LR were observed. The alterations noted when hepatocytes were exposed to 50 micrograms/L MC-LR were blebbing of cell membrane, shrinking and deformation of nuclei, vesiculation and transformation into concentric membrane whorls of RER, and swelling and rearrangement of the cytoskeleton. However, when the cells were exposed to 500 micrograms/L MC-LR, broken cell membranes, nuclei and cytoskeleton could be observed. These ultrastructural changes paralleled the pathological events, which lead to apoptosis or necrosis of hepatocytes. These results suggest that disruption of the cytoskeletal structures could account for the blebbing of cell membrane and apoptosis induced by MC-LR.