Temperature effects on the development of Enteromyxum spp. (Myxozoa) in experimentally infected tiger puffer, Takifugu rubripes (Temminck & Schlegel).

The effects of water temperature on the development of the enteric myxosporeans, Enteromyxum fugu and Enteromyxum leei, were investigated in experimentally infected tiger puffer, Takifugu rubripes. After naïve tiger puffer were fed gut tissue infected with both E. fugu and E. leei, they were divided into separate tanks and kept at different constant temperature regimes between 10 and 25 degrees C. Regardless of the water temperature tested, E. fugu was consistently detected with a high prevalence of infection (60-100%), although no sporulation occurred at 10 and 15 degrees C. Development of E. leei and the onset of disease were suppressed by low water temperatures (<15 degrees C). However, a temperature increase to 20 degrees C promoted the development of E. leei, followed by an increase of disease rate in the fish. The present study demonstrates that water temperatures below 15 degrees C have an inhibitory effect on the development of E. fugu and E. leei, resulting in suppression of enteromyxosis at low temperatures.

Clinicopathological correlation of polypoidal choroidal vasculopathy revealed by ultrastructural study.

AIMS: To describe the clinical and histopathological findings in a patient with polypoidal choroidal vasculopathy. METHODS: A 76 year old Japanese man had a discrete, orange-red lesion of 1 disc diameter in the macula, with the fluorescein and indocyanine green angiographic and optical coherence tomographic findings compatible with polypoidal choroidal vasculopathy. He underwent a surgical removal of the macular lesion, followed by light and electron microscopic examinations. RESULTS: The histopathological examination revealed that the specimen consisted of degenerated retinal pigment epithelium-Bruch's membrane-choriocapillaris complex and inner choroid. A tortuous, unusually dilated venule was present adjacent to an arteriole with marked sclerotic changes, appearing to form arteriovenous crossing. These vessels seemed to represent native inner choroidal vessels, and had haemorrhage per diapedesis. Blood cells and fibrin filled the lumina of the vessels and accumulated in the extravascular spaces, indicating vascular stasis. CONCLUSION: Hyperpermeability and haemorrhage due to stasis of a dilated venule and an arteriole involved by sclerosis at the site where they cross in the inner choroid might cause oedema and degeneration of the tissue. Voluminous accumulation of blood cells and fibrin might generate elevation of tissue pressure sufficient to displace the weakened lesion anteriorly. The result suggests that the polypoidal vessels in this case represent abnormality in the inner choroidal vasculature.

S-Adenosyl-L-homocysteine hydrolase is sequestered into actin rods in Dictyostelium discoideum spores.

Here we show evidence that S-adenosyl-L-homocysteine hydrolase (SAHH) is linked to the actin cytoskeleton. Actin rods formed in Dictyostelium discoideum spores during the final stage of development are structurally composed of novel bundles of actin filaments. SAHH only accumulates with actin at this stage of development in the life cycle of D. discoideum. Recently SAHH is believed to be a target for antiviral chemotherapy and the suppression of T cells. Our finding may contribute to designing novel antiviral and immunosuppressive drugs.

Cofilin-2, a novel type of cofilin, is expressed specifically at aggregation stage of Dictyostelium discoideum development.

BACKGROUND: A conventional cofilin, cofilin-1 in Dictyostelium discoideum plays significant roles in cell proliferation, phagocytosis, chemotactic movement and macropinocytosis. RESULTS: We identified a new member of the cofilin family, named cofilin-2 in D. discoideum. Cofilin-2 shows significant homology to a conventional Dictyostelium cofilin, cofilin-1, through its entire sequence, and contains residues conserved among the cofilin family that are responsible for actin-binding. On the other hand, several residues that are conserved among the cofilin family are missing from cofilin-2. Purified cofilin-2 depolymerized actin filaments in a dose- and pH-dependent manner and reduced the apparent viscosity of an actin solution, although they did not co-sediment with actin filaments at all. Cofilin-2 was not expressed in vegetative cells, but was transiently induced during the aggregation stage of development, whereas cofilin-1 was predominantly expressed in vegetative cells. Immunocytochemistry revealed that cofilin-2 localizes at substrate adhesion sites, where cofilin-1 is almost completely excluded. Disruption of the cofilin-2 gene caused an increase in actin accumulation at the substrate adhesion sites. We also found that cofilin-2 did not rescue Deltacof1 yeast cells, whereas cofilin-1 did. CONCLUSIONS: Cofilin-2 may play a distinct role from that of cofilin-1 in destabilization of the actin cytoskeleton during Dictyostelium development.

The formation of actin rods composed of actin tubules in Dictyostelium discoideum spores.

A new type of actin rod formed in both the nucleus and the cytoplasm, as well as tyrosine phosphorylation of actin, is implicated in the maintenance of dormancy and viability of Dictyostelium discoideum spores. Here the ultrastructure of the rods and their relationship to the phosphorylation of actin were examined. The rods first appeared in premature spores at the midculmination stage as bundles composed of actin tubules hexagonally cross-linked. The 13-nm-diameter bundles were composed of three actin filaments. Formation of the actin rods begins during the late culmination stage and proceeds until 2 days after completion of fruiting bodies. The physical events occur in the following order; association of several modules of bundles, close packing and decrease in diameter of actin tubules, elongation of rods across the nucleus or the cytoplasm. Actin phosphorylation levels increased at the late culmination stage and reached a maximum level 12 h later. Immediately following activation of spore germination, actin was rapidly dephosphorylated, followed shortly thereafter by the disappearance of rods. Shortened actin tubules once again became arranged in a hexagonal pattern. This hexagonal arrangement of actin tubules is possibly involved in rod formation and disappearance and does not depend upon actin phosphorylation. In contrast, rod-maturation processes may correlate with actin phosphorylation.

End-linked homodimers in fibrinogen Osaka VI with a B beta-chain extension lead to fragile clot structure.

The authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bbeta gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient. (Blood. 2000;96:3779-3785)

Ultrastructural changes associated with accumulation of inclusion bodies in rat retinal pigment epithelium.

PURPOSE: To determine the structural changes in the retinal pigment epithelium (RPE) and neighboring structures induced by intravitreal injection of a lysosomal protease inhibitor. METHODS: Eleven-week-old Sprague-Dawley rats were injected with 5 microliter of a lysosomal protease inhibitor, E-64 (2.22 microM), intravitreally once and killed at 24 hours, 48 hours, or 7 days later. Others received two or three injections at 48-hour intervals or three daily injections, and killed at 1, 4, and 7 days after the last injection. Eyes were enucleated and retinal tissues were processed for light and electron microscopy. RESULTS: A single injection of E-64 caused only a transient accumulation of phagosome-like and phagolysosome-like inclusion bodies in the RPE. By contrast, repeated injection caused progressive accumulation of these inclusions followed by altered RPE cell conformation, and changes in organelles such as loss of smooth endoplasmic reticulum (SER). This was accompanied by shortening and loss of photoreceptor outer segments without prior dysmorphic changes, alteration of choroidal capillaries, and invasion of Bruch's membrane by fibroblasts and pericytes. Intravitreal injection of vehicle as control induced no structural changes. CONCLUSIONS: E-64 treatment induced structural changes in the outer retina. The causal relationship between accumulation of inclusions in RPE and changes in other subcellular organelles and neighboring cells systems is not clear. However, there are possible explanations: physical disturbance of organelles, particularly SER by inclusions; cellular damage by consequent upon accumulation of A2-E; or, shortage of recycled material due to reduced degradation of phagosomes.

Glucose-induced pathways for actin tyrosine dephosphorylation during Dictyostelium spore germination.

In the presence of germination signals, dormant spores of Dictyostelium discoideum rapidly germinate to start a new life cycle. Previously we have shown that half of the actin molecules in spores are maintained in a tyrosine-phosphorylated state, and a decline of the actin phosphorylation levels is a prerequisite for spore swelling. In this study, we have established d-glucose as a trigger molecule for the actin dephosphorylation. Present in a nutrient germination medium, d-glucose both may act as a trigger molecule and/or may serve as a substrate within a pathway for actin dephosphorylation depending upon spore age. However, the glucose-induced actin dephosphorylation was insufficient for spores to swell. Other factors in the nutrient medium were required for complete germination of young spores aged 1 to 5 days. In contrast, dispersion in nonnutrient buffer was necessary and sufficient for a decline of actin phosphorylation levels and even the emergence of amoebae in older spores (6 days and beyond). Moreover, the dephosphorylation pathway in the older spores was independent of energy production. We propose that the diversification of the actin dephosphorylation pathway may enable spores to increase their probability of germination upon spore aging.

Novel actin cytoskeleton: actin tubules.

In spores of Dictyostelium discoideum three actin filaments are bundled to form a novel tubular structure and the tubules are then organized into rods. These tubular structures we will term actin tubules. Actin tubules are reconstructed from the supernatant of spore homogenates, while the usual actin filaments were bundled after incubation of supernatants from growing cells. Alpha-actinin, ABP-120 and EF-1alpha are not essential for rod formation. Cofilin is a component of the cytoplasmic rods but few cofilin molecules are included in the nuclear rods. The viability of spores lacking actin rods is very low, and the spore shape is round instead of capsular. The rods can be fragmented by pressure, indicating that the rods may be effective in absorbing physical pressure. The complex organization of actin filaments, actin tubules and rods may be required for spores to achieve complete dormancy and maintain viability.

Hyperosmotic stress-induced reorganization of actin bundles in Dictyostelium cells over-expressing cofilin.

BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin-interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature-sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known. RESULTS: In response to hyperosmotic stress, D. discoideum cells round up. This stress-induced morphological change involves the redistribution of cofilin, together with actin filaments, into cortical contractile portions of the cells, followed by their contraction. Over-expression of cofilin increases and thickens cortical actin bundles in cells. The bundles become tight and are reorganized into a ring-shaped structure in response to hyperosmotic stress. The ring structure of actin bundles had two characteristic bands across them; bright and dark bands, heavily stained and not stained with phalloidin. In the bundles, straight filaments with a diameter of 5.3-nm were aligned parallel by cross-bridge structures. In cells lacking the myosin-II heavy chain, the bundles, which were induced by an over-expression of cofilin, shortened and became straight following hyperosmotic stress, forming a polygonal structure. D. discoideum Aip1/Wrp2 enhanced the severing of actin filaments by cofilin in vitro and colocalized with cofilin in cells, including those that were over-expressing cofilin before and after exposure to hyperosmotic stress. CONCLUSIONS: Cofilin plays a pivotal role in concert with Aip1/Wrp2 in the reorganization of actin architectures into bundles that contract in a myosin-II-independent manner, in response to hyperosmotic stress.

Monomer arrangement in HSP90 dimer as determined by decoration with N and C-terminal region specific antibodies.

Electron microscopy using the low-angle rotary shadowing replica method showed that the HSP90 dimer consists of four globular domains aligning in a tandem fashion. When decorated with two monoclonal antibodies against epitopes mapped on the N-terminal region of HSP90, these antibodies bound to both ends of the HSP90 dimer. A C-terminal region specific antibody was shown to bind to the side of HSP90. These results support a model for HSP90 dimer whereby two HSP90 monomers are arranged in an antiparallel fashion and dimerize through the C-terminal domain. Treatment of HSP90 at elevated temperatures or with ATP at room temperature, though not with ADP, induces molecular transformation of the linear HSP90 dimer into an O-ring-shaped structure.

Ultracytochemical demonstration of glycogen in cone, but not in rod, photoreceptor cells in the rat retina.

The presence of native glycogen in photoreceptor cells of the rat retina has not been identified in the literature. We have studied this ultracytochemically. After perfusion with glutaraldehyde fixative, the eyes were enucleated, and the retinal tissues, postfixed with OsO4, were embedded in epoxy resin. Some tissues were treated with saliva before postfixation. Ultrathin sections, stained by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method or with uranyl acetate and lead citrate, were examined by electron microscopy. On routinely stained sections, glycogen particles seemed to be absent in the cytoplasmic matrix of the photoreceptor cells because they were indistinguishable from the numerous ribosomes. This was due to a similarity in size and electron density. After PA-TCH-SP staining, fine electron-dense reaction products appeared on small cytoplasmic particles (but not on ribosomes) in the inner segments, perikarya and synaptic terminals of a subpopulation of photoreceptor cells. These particles, 15-25 nm in diameter, were identified as beta-particles of glycogen because of their susceptibility to enzyme digestion. The glycogen-rich photoreceptor cells were thought to be cone cells by reasons of their morphological features, such as synaptic terminals, nuclei and outer segments. These results suggest that the cone, but not the rod, photoreceptor cells in the rat contain abundant glycogen.

High levels of actin tyrosine phosphorylation: correlation with the dormant state of Dictyostelium spores.

Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days. Spores having high levels of phosphorylation show high viability, and vice versa. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process.

Gray-white, spherical deposition on retinal vessel associated with acute retinal necrosis and diabetic retinopathy in HTLV-I carriers.

Tiny, gray-white, spherical deposits were found on the retinal vessels and vitreoretinal interface of the fovea in a 55-year-old woman with acute retinal necrosis due to varicella-zoster virus, and in a 47-year-old man with preproliferative diabetic retinopathy. Both patients developed massive vitreous opacities due to uveal inflammation or hemorrhages that rendered the fundus difficult to visualize. A therapeutic vitrectomy revealed gray-white, tiny spherical deposits, about blood-vessel-diameter, which were scattered on seemingly intact retinal arteries and veins in the posterior fundus and over the vitreoretinal interface overlying the fovea. The deposits were loosely adherent to vessel walls and were easily aspirated, and the residual materials resolved in the early postoperative days. These characteristic retinal vascular deposits resembled those seen in patients with HTLV-I associated uveitis. The two patients reported herein were otherwise asymptomatic carriers of HTLV-I. The findings provide additional information about the etiological role of HTLV-I in the development of characteristic retinal vascular deposition, although its pathogenesis remains to be elucidated.

[Selective binding of fucose-recognizing lectin on the cone photoreceptor outer segments].

The distribution of fucose-containing glycoconjugates in the photoreceptor cell layer of rat and human retinas was examined by lectin histochemistry using Aleuria aurantia lectin (AAL), which recognizes L-fucose alpha 1, 6 residue. In the rate retina, AAL diffusely bound to the apical outer segments and to the basal inner segments, whereas it bound to the entire outer segments of other photoreceptors, which were considered to be cones due to their proportion. In the human retina, AAL bound diffusely to the basal inner segments and to the retinal pigment epithelia, but it bound selectively to the outer segments of the cones. The present findings revealed that the glycoconjugates, whose sugar chains contain L-fucose alpha 1, 6 residue on their termini, are present in the cone outer segments.

A green fluorescent protein-actin fusion protein dominantly inhibits cytokinesis, cell spreading, and locomotion in Dictyostelium.

We transformed Dictyostelium discoideum cells by a vector for expression of a chimerical fusion protein consisting of Aequorea Victoria green fluorescent protein (GFP) and D. discoideum actin at its amino- and carboxy-terminal, respectively. The amount of expressed GFP-actin was about 3% of total actin molecules in the transformed cells. The expression of GFP-actin in D. discoideum completely inhibited cytokinesis in suspension culture. The expression decreased the rate of random cell locomotion to about a half of that of control cells. The expression also caused the cells to round up. These phenotypic observations suggested that GFP-actin acts as a dominant negative form of actin in the cells. The rounding up by expression of GFP-actin was suppressed by genetical elimination of myosin II heavy chain. This result suggested that myosin II is necessary for the rounding up of GFP-actin expressing cells. GFP-actin constructed cortical actin filament architectures together with intrinsic actin in the cells. Purified GFP-actin polymerized and de-polymerized repetitively according to the solution conditions in vitro. The critical concentration of GFP-actin for polymerization is the same as that of actin. The GFP-actin filaments was able to bind to coverglass surfaces coated with myosin head fragments. However, the GFP-actin filaments did not slide at all on the coverglass by addition of ATP. This indicates that the GFP-actin filaments form rigor complex with myosin II in vitro even in the presence of ATP. The formation of rigor complex may cause the cells to round up.

Immunohistochemical localization of cytosolic sialidase in photoreceptor cells.

The binding sites of the antibody to cytosolic sialidase on the rat and monkey photoreceptor cells were examined immunohistochemically using the avidin-biotinylated peroxidase method. In the rat photoreceptor cells, the antibody bound diffusely to the inner segment and the outer nuclear layers which are composed chiefly of rod cells. In the monkey photoreceptor cells, the antibody bound to the rod inner segments which were clearly distinct, morphologically, from the cone inner segments. The antibody also bound to the rod cell bodies in the outer nuclear layer. These binding patterns show that the antibody bound preferentially to rod photoreceptor cells. This observation is consistent with previous lectin histochemical findings that sialoglycans are preferentially present on the surfaces of rod photoreceptors, and in the rod-associated interphotoreceptor matrix. Sialidase in rod inner segments may function by balancing with sialyltransferase, also preferentially expressed in rod inner segments, to form sialyl residues on the termini of sugar chains in the rod-associated sialoglycoconjugates.

Lectin-histochemical study of O-linked glycoconjugates in dysplastic retina of Norrie disease.

The carbohydrate chains of O-linked glycoconjugates in dysplastic retina from a Japanese female infant with Norrie disease were examined by lectin histochemistry. The avidin-biotinylated peroxidase method was used. The retina was highly dysplastic and composed of undifferentiated embryonic tissues containing a number of rosettes of varying sizes. The lumina of the rosettes were stained by peanut agglutinin, which recognizes the Gal beta 1,3GalNAc sequence of O-linked glycans. However, the lumina were not labeled by wheat germ agglutinin, which reacts with sialic acid and/or N-acetylglucosamine. These observations suggest that the O-linked glycoconjugates in the lumina of rosettes were not sialylated in the present case. Their lack of terminal sialic acids may be related to the rosette formation.