ATP-dependent structural changes of the outer dynein arm in Tetrahymena cilia: a freeze-etch replica study.

With the rapid-freeze, deep-etch replica technique, the structural conformations of outer dynein arms in demembranated cilia from Tetrahymena were analyzed under two different conditions, i.e., in the absence of ATP and in the presence of ATP and vanadate. In the absence of ATP, the lateral view of axonemes was characterized by the egg-shaped outer dynein arms, which showed a slightly baseward tilt with a mean inclination of 11.1 degrees +/- 3.4 degrees SD from the perpendicular to the doublet microtubules. On the other hand, in the presence of 1 mM ATP and 100 microM vanadate, the outer arms were extended and slender and showed an increased baseward tilt with a mean inclination of 31.6 degrees +/- 4.9 degrees SD. In ATP-activated axonemes, these two types of arms coexisted, each type occurring in groups along one row of outer arms. These findings strongly suggest that the interdoublet sliding is caused by dynamic structural changes of dynein arms that follow the hydrolysis of ATP.

Myosin filaments in smooth muscle cells of the guinea pig taenia coli: a freeze-substitution study.

Myosin filaments in smooth muscle cells of the guinea pig taenia coli were studied by freeze-substitution electron microscopy combined with rapid freezing using liquid helium. The thick myosin filaments were reproducibly observed both in relaxed and contracted states. In relaxed states, the thick filaments are more or less lined up among the packed bundles of thin filaments. The basic arrangement and relative ratio of thick and thin filaments were the same between the relaxed and contracted cells, except that many of thick filaments were often observed to be very close to thin filaments in contracted states. The results indicate that myosin in smooth muscle cells exists as thick filaments at any stages during contraction-relaxation cycle.

Complete suppresion of ethanol-induced formation of megamitochondria by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO).

An attempt has been made to suppress the ethanol-induced formation of megamitochondria (MG) in the rat liver by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO), a free radical scavenger, and by allopurinol (AP), a xanthine oxidase inhibitor. Changes observed in the liver of animals given ethanol (EtOH) for 1 month were remarkable decreases both in the body weight gains during the course of the experiment and in the liver weight at the time of sacrifice compared to those of the control; remarkable increases in the level of thiobarbituric acid reactive substances and lipid soluble fluorophores both in microsomes and mitochondria; decreases in the content of cytochrome a+a3 and b and lowered phosphorylating ability of mitochondria; and formation of MG in the liver. A combined treatment of animals with EtOH plus 4-OH-TEMPO completely suppressed the formation of MG in the liver induced by EtOH and distinctly improved the changes caused by EtOH, as specified above, while AP partly suppressed the MG formation. Results described herein provide additional insight into chronic hepatotoxicity of EtOH besides that previously reported. A novelty of the present work is that we were able for the first time to demonstrate reversibility of EtOH-mediated ultrastructural changes of the liver by a simple administration of aminoxyl-type free radical scavenger, 4-OH-TEMPO. Our results suggest that free radicals may be involved in the mechanism of the formation of MG induced by EtOH.

Free radical-induced megamitochondria formation and apoptosis.

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O2- from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.

The cytoskeleton in myelinated axons: a freeze-etch replica study.

The organization of the cytoskeleton in myelinated axons of the rat has been analyzed without chemical fixation in replicas of deep-etched materials after rapid freezing. Freeze-etch replicas of trigeminal nerves provided three-dimensional views of the well-developed cytoskeleton inside axons. In these preparations, the axonal cytoskeleton was seen to be composed of longitudinally-oriented microtubules and neurofilaments which were interconnected by slender strands. Such strands also connected membranous organelles with microtubules and neurofilaments. After Triton X-100 extraction, the neurofilament-associated interconnecting strands (cross-linking filaments) persisted, indicating that they are not artifactual products of soluble protein condensation during freeze-etching. In non-extracted axons many granular structures were closely associated with cytoskeletal components. These granular elements were not seen after Triton treatment. These findings, together with fluorographic analyses, suggest that the granular structures may represent. These findings, together with fluorographic analyses, suggest that the granular structures may represent slowly transported "soluble proteins' in axoplasm. This freeze-etch replica study, without any chemical fixation, substantiates the reality of the axonal cytoskeleton which is directly involved in the axonal transport. Furthermore, using this approach ultrastructural evidence was obtained of the close association of membranous structures with the cytoskeleton.

Localization of caveolin-1 in photoreceptor synaptic ribbons.

PURPOSE: The purpose of this study is to determine whether caveolin-1 is a constituent of photoreceptor synaptic ribbons. METHODS: Immunoblot assay and electron microscopic immunocytochemistry were used to localize caveolin-1 in synaptic ribbons. RESULTS: Synaptic ribbons were localized close to the active site of presynaptic membranes and surrounded by a halo of synaptic vesicles. Immunosignals of caveolin-1 were clearly detected on the synaptic ribbons in rod and cone photoreceptors. However, the signal was seen neither on synaptic vesicles nor on presynaptic plasma membranes. CONCLUSIONS: Caveolin-1 is a component protein of synaptic ribbons and may be involved in the regulation of transmitter release.

[3H]ouabain localization of Na-K ATPase in the epithelium of rabbit ciliary body pars plicata.

The secretion of the aqueous humor has been proposed to occur as the result of active Na+ transport by a ouabain-sensitive Na-K ATPase. We have examined the localization of this enzyme in the epithelium of rabbit ciliary body pars plicata using [3H]ouabain autoradiography. Single ciliary processes were isolated and incubated in Ringer containing [3H]ouabain. Processes were then rapidly frozen, freezedried, sectioned and exposed for autoradiography. In the light microscope, silver grains were found predominantly over the nonpigmented epithelial cells. In the electron microscope, grains could be localized for the most part to the interdigitations of the nonpigmented cell basolateral membrane. Label could also be observed at a much lower density above other membranes and above the pigmented and nonpigmented cell cytoplasm. No label was found in sections of control tissue which had been incubated in [3H]ouabain with an excess of cold ouabain. To show that the [3H]ouabain had free access to all of the membrane surfaces within the epithelium, in parallel experiments we incubated isolated processes in horseradish peroxidase. Our experiments suggest that most of the active Na+ transport in ciliary body epithelium occurs across the basolateral membrane of nonpigmented cells into the posterior chamber. Furthermore, the placement of the Na-K ATPase within the narrow membrane infoldings of the interdigitations is consistent with a role for this enzyme in water transport and the production of the aqueous.

Retinal dysfunction in basigin deficiency.

PURPOSE: To examine the retina of basigin (Bsg) knockout mice by electrophysiological and histologic methods and thereby to determine the possible function of Bsg in phototransduction and retinal development. METHODS: Scotopic and photopic electroretinograms (ERGs) were recorded from 11 wild-type, 12 heterozygous, and 8 homozygous Bsg gene knockout mice of different ages. The retinas were also examined by histologic and immunolabeling methods. RESULTS: Bsg knockout mice of 5 to 41 weeks of age showed a decrease in the amplitude of all components of both the photopic and scotopic ERGs. In contrast, the fundus and the fluorescein fundus angiography and morphology of the retina at the light microscopic level appeared to be normal until 8 weeks of age in Bsg knockout mice. Thereafter, the length of outer segment and outer nuclear layers decreased with increasing age. Immunohistochemical analysis localized Bsg protein in a variety of cells in the retina, especially in the pigment epithelium, the upper outer plexiform layer and the inner segments of photoreceptor cells. CONCLUSIONS: The results demonstrated that both rod and cone function were severely affected from an early age by the targeted disruption of the Bsg gene. In spite of abnormal ERGs, the photoreceptor cells maintained normal morphology up to 8 weeks. Thereafter, the photoreceptor cells degenerated gradually and were almost ablated by 41 weeks.

Studies on urea synthesis in the liver of rats treated chronically with ethanol using perfused livers, isolated hepatocytes, and mitochondria.

Changes in urea synthesis in the liver of rats treated with 32% ethanol in the drinking water for up to 6 months were studied using perfused livers, isolated hepatocytes, and mitochondria. Results obtained from ethanol-treated rats are summarized as follows: (1) the mitochondria of the hepatocytes of rats treated with ethanol for 2 months or longer became enlarged to various degrees, (2) the levels of ammonia in the serum remained within a normal range, while those in liver tissue were elevated compared with the control, (3) urea synthesis from ammonia in perfused livers was decreased markedly, while that from citrulline remained in the normal range, (4) the activities of carbamyl phosphate synthetase (CPS; EC 2.7.2.5) and ornithine transcarbamylase (OTC; EC 2.1.3.3) in mitochondria were unchanged compared with those of the control, and (5) the levels of ATP in liver tissue and the ability of mitochondria to synthesize ATP were decreased markedly compared with the control. Both the level of ATP in the hepatocytes and the synthesis of urea from ammonia by perfused livers of rats treated with ethanol were resistant to externally added ethanol, while those of control animals were severely affected. These results suggest that the intracellular level of ATP is intimately related to urea synthesis in both control and ethanol-treated animals, and lowered levels of ATP may be a key factor in the suppression of urea synthesis in ethanol-treated animals.

Phosrestin I, an arrestin homolog that undergoes light-induced phosphorylation in dipteran photoreceptors.

Two classes of phosphorylated homologs of vertebrate arrestins, designated phosrestins I (PRI) and phosrestin II (PRII), are expressed in the photoreceptors of a fruit fly, Drosophila melanogaster. This study presents evidence that the housefly, Musca domestica, also has a protein similar to Drosophila PRI. Our conclusion is based on the following evidence. (1) We identified a Musca photoreceptor protein exhibiting a molecular mass (51 kDa) and an isoelectric point (pI = 8.6) similar to those of Drosophila PRI. This Musca protein, designated Musca PRI, changes its pI upon illumination in vivo. Drosophila PRI. This Musca protein, designated Musca PRI, changes its pI upon illumination in vivo. (2) Rabbit antibodies raised against Musca PRI, against bovine arrestin, and against a synthetic peptide based on the Drosophila PRI sequence stained the Drosophila and Musca PRIs specifically on 1 and 2-dimensional Western immunoblots. (3) Both Drosophila and Musca PRIs incorporated 32P-radioactivity from gamma-32P-ATP in cell-free homogenates of retinas. Partial peptide digestions of Drosophila and Musca PRIs revealed similarity between these proteins. We observed that Drosophila PRI exists in the random preparation, but it also exists in other subcellular fractions. Immunocytochemistry at the EM level revealed a distribution of both Drosophila and Musca PRI epitopes in membranous vesicular structures in the cytosol as well as in the rhabdomeric microvillar membranes where the visual pigment, rhodopsin, exists. Such distribution of PRI epitopes suggests that PRI and its light-dependent phosphorylation may function in a space remote from the rhabdomere as well as the immediate milieu of photoreception.

A freeze-fracture study of the aggregation state of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum in reconstituted vesicles at low and high temperature.

Since it was possible for Ca2+,Mg2+-ATPase of sarcoplasmic reticulum (SR) to change its aggregation state in the membrane depending on temperature, and since the change could be the cause of the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity, the aggregation state of Ca2+,Mg2+-ATPase at 0 degrees C in the membrane was compared with that at 35 degrees C by freeze-fracture electron microscopy. These temperatures are below and above the break in the Arrhenius plot (about 18 degrees C), respectively. Two kinds of samples were used; fragmented SR vesicles and egg PC-ATPase vesicles, a reconstituted preparation from purified Ca2+,Mg2+-ATPase and egg yolk phosphatidylcholine (egg PC). For both the appearance of particles in the fracture faces of the samples fixed at 0 degrees C was similar to that at 35 degrees C, and phase separation between protein and lipid was not observed even at 0 degrees C. The size of the particles was measured and histograms of the sizes at 0 degrees C and 35 degrees C were made. The histogram at 0 degrees C was similar to that at 35 degrees C with a peak at 7.1 nm, which is 1-2 nm smaller than the value reported so far. The number of the particles per unit area of the membrane was also counted. The value at 0 degrees C was similar to that at 35 degrees C. These results indicate that Ca2+,Mg2+-ATPase of SR exists in the same aggregation state (estimated as oligomer based on the values obtained in this experiment) between 0 degrees C and 35 degrees C. Based on the results of this study we think that the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity in SR is not caused by the change in the aggregation state of Ca2+,Mg2+-ATPase.

Mechanism of the formation of megamitochondria in the mouse liver induced by chloramphenicol.

Correlation between chloramphenicol-induced formation of megamitochondria in the mouse liver and oxidative stress was studied by lipid peroxidation analysis and electron microscopic technique. Chloramphenicol suppressed increases in the body weight and liver weight of experimental animals and at the same time induced a remarkable increase in lipid peroxidation in the liver during the formation of megamitochondria. A spin trapping agent, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, abolished all these changes induced by chloramphenicol. Namely, both the body weight and liver weight of chloramphenicol-treated animals stayed at the same levels as those of the control, and the formation of megamitochondria was completely suppressed. Allopurinol, a xanthine oxidase (EC 1.2.3.2) inhibitor, partly inhibited the changes induced by chloramphenicol, as described above. These results suggest that chloramphenicol-induced formation of megamitochondria is not simply ascribed to the suppression of the dividing process of mitochondria due to lowered protein synthesis in mitochondria but is intimately related to oxidative stress. Furthermore, the results obtained with allopurinol may indicate that enhanced levels of lipid peroxidation observed in chloramphenicol-treated animals are partly due to enhanced rate of the degradation of purine nucleotides catalyzed by xanthine oxidase.

Effect of lipid emulsions for total parenteral nutrition on regeneration of the liver after partial hepatectomy in rats.

We studied the effects of lipid emulsions for total parenteral nutrition (TPN) on hepatic regeneration after partial hepatectomy in rats. Daily energy intake was maintained at 1172 kJ.kg-1.day-1 while the percentage of nonprotein energy sources was changed. Animals were divided into four groups: lipid-free, 10%-lipid, 20%-lipid, and 40%-lipid. TPN was continued for up to 1 wk. The content of proteins, the ratio of proteins to triglycerides, and the yield of mitochondrial protein in the remnant liver 7 days after partial hepatectomy were larger in animals receiving TPN with lipids than in those receiving lipid-free TPN, whereas the amounts of triglycerides and cholesterol in the liver of the latter animals were larger. The degree of fatty infiltration of the hepatic lobule was most distinct in the lipid-free group. Furthermore, activities of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and alkaline phosphatase in the serum tended to be higher in the lipid-free group. Phosphorylating ability of mitochondria in the regenerating liver 7 days after partial hepatectomy was not different among the four groups; however, the highest value for the respiratory control index was obtained in the 40%-lipid group. The application of a lipid emulsion to TPN is useful for hepatic regeneration after partial hepatectomy; however, the ideal concentration of lipids in TPN awaits further investigation.

Molecular mimicry: uveitis induced in Macaca fascicularis by microbial protein having sequence homology with retinal S-antigen.

S-antigen (S-Ag), a well characterized 45-kDa protein in the photoreceptor cells, induces predominantly T-cell-mediated autoimmune uveitis when injected into experimental animals. Recently, we have shown that native histone H3 protein derived from yeast (Saccharomyces cerevisiae), or a synthetic peptide that is homologous with S-Ag peptide M in having six consecutive amino acids, induces experimental autoimmune uveitis (EAU) similar to that induced by native S-Ag in the Lewis rat. In this study, monkeys (Macaca fascicularis) immunized with histone H3 peptide developed a strong cellular immune response to this peptide as well as to peptide M. However, no significant inflammation or hypervascularization was observed in the retina or the iris during the experimental period, when they were examined clinically with an inverted ophthalmoscope. Histopathological examination showed that all monkeys injected with histone H3 peptide or with native histone H3 lost a large number of photoreceptor rod cells and developed neovascularization in the outer nuclear cell layer of the retina. These histopathological findings in the monkey retina closely resemble those seen in human patients with some types of uveitis. The possible involvement of microbial proteins having sequence homology with normal retinal proteins in the pathogenicity of human uveitis is discussed.

S-antigen localization in developing rds mouse retina.

The morphology of the photoreceptor cell and localization of the cytoplasmic soluble protein, S-antigen, in the retina of the developing retinal degeneration slow (rds) mutant mouse (2-505 postnatal days) were studied by improved immunocytochemical and freeze-substitution methods. Anti-S-antigen antibody labeling was observed first in the postnatal 10-day retina under light microscope. Labeling signals increased progressively to a maximum level in 20 days, and then decreased gradually to an undetectable level by 505 postnatal days. By electron microscopic immunocytochemical methods, S-antigen was detected first in the photoreceptor cells at 3 postnatal days, and increased with development. It was located in the entire cytoplasm of the photoreceptor cell including the rudimentary outer segment, but degenerated and disappeared by 505 postnatal days. S-antigen was also present in the membranous vesicles budding off from the photoreceptor membrane to the subretinal space. The rds photoreceptor cell seems to lose other soluble proteins together with these vesicles. From these results and other published data, we speculate that the degeneration of the photoreceptor cells may be the secondary effects of the loss of a large amount of soluble and membrane proteins following the malfunction of membrane. Recent reports show the rds mouse must have a gene defect in the membrane component such as peripherin or the 39 kDa protein.

Image analysis of electron micrographs of ATPase (Coupling Factor TF1) from thermophilic bacteria and luminal epithelium of mouse urinary bladder.

Electron micrographs of two dimensional array of H+-ATPase (Coupling Factor TF1 from the thermophilic bacteria) and luminal epithelial protein of urinary bladder were obtained using negative staining method and freeze-fracture method, respectively. Images which showed good optical diffraction pattern were digitized by a computer-linked flat-bed two dimensional microdensitometer and processed digitally. The results of translational computer noise filtering showed that the outline of TF1 molecules looks like a hexagon or an asterisk in the presence of sodium azide which is a specific inhibitor of TF1 or AMPPNP which is an unsplitable analogue of ATP, respectively. The luminal epithelial protein also looks like a hexagon. Rotational harmonic analysis was carried out to examine the rotational symmetry of the filtered images of TF1. It was found that 2-fold or 6-fold symmetry is dominant in the presence of sodium azide or AMPPNP, respectively.

Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells.

Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions.

Morphogenesis of the photoreceptor outer segment during postnatal development in the mouse (BALB/c) retina.

Disc formation of rod photoreceptor cells in developing BALB/c mice retinas was studied by rapid freeze, freeze-substitution, freeze-etching, immunocytochemistry, and myosin S-1 decoration methods. Freeze-substituted photoreceptor cells contained variously shaped vesicles in the apical swelling of the connecting cilium or the base of the outer segment during postnatal development. Rapid freezing successfully arrested pinocytosis; the fusion of small vesicles to give large ones, and the compression of certain vesicles (0.3-0.6 micron) appears to lead gradually to the formation of the so-called discs. We therefore propose that membranous discs are formed by the fusion of small pinocytotic vesicles and their subsequent compression. Discs formed in this way were partially stacked, but were ordered at random during the early developmental stages. During development, a partial stack of discs was progressively rearranged to a regular form as seen in mature outer segments. Cytoskeletal actin was expected to be involved in the disc formation; it was demonstrated in the distal axoneme of the connecting cilium during development and showed no change in its distribution. However, the polarity of the actin filaments, as revealed by myosin S-1 decoration in early developmental stages, was much more variable than in the adult. Barbed ends of actin filaments were associated with the plasma membrane or the membrane of vesicles. We also found actin filaments coiled up helically on ciliary microtubules.