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.

Spatial and temporal dynamics of two alternatively spliced regulatory factors, lens epithelium-derived growth factor (ledgf/p75) and p52, in the nucleus.

Regulatory factors, lens epithelium-derived growth factor (LEDGF)/p75 and p52, are generated from a single LEDGF gene by alternative splicing. They have identical amino acid residues between positions 1-325, but 205 and 8 of the remaining residues are different in LEDGF and p52, respectively. LEDGF promotes growth and survival of many cell types. It has an antiapoptotic function and is a weak general transcriptional co-activator. p52 is a transcriptional activator and an essential splicing factor. We investigated the spatial and temporal dynamics of LEDGF/p75 and p52, each being tagged with a fluorescent protein, during the cell cycles of CHO-K1, MCDK, and NRK cells in culture. Both LEDGF/p75 and p52 were localized predominantly in the nucleus. LEDGF/p75 was distributed diffusely in the nucleoplasm in the G1-phase and attached to chromatin heterogeneously during the G2 and M-phases of cells. In contrast, p52 was localized in the nuclear periphery during the G1-phase and formed a speckle pattern at the S-phase. It formed a cylindrical pattern around the chromosomes during the M-phases of cells. LEDGF and p52 on sister chromatids migrated into daughter cells. Thus, LEDGF/p75 and p52 are localized in distinct nuclear compartments where they can activate transcription or splicing of pre-mRNAs.

Physical interactions between DinI and RecA nucleoprotein filament for the regulation of SOS mutagenesis.

The Escherichia coli dinI gene is one of the LexA-regulated genes, which are induced upon DNA damage. Its overexpression conferred severe UV sensitivity on wild-type cells and resulted in the inhibition of LexA and UmuD processing, reactions that are normally dependent on activated RecA in a complex with single-stranded (ss)DNA. Here, we study the mechanism by which DinI inhibits the activities of RecA. While DinI neither binds to ssDNA nor prevents the formation of RecA nucleoprotein filament, it binds to active RecA filament, thereby inhibiting its coprotease activity but not the ATPase activity. Furthermore, even under in vitro conditions where UmuD cleavage dependent on RecA-ssDNA-adeno sine-5'-(3-thiotriphosphate) is blocked in the presence of DinI, LexA is cleaved normally. This result, taken together with electron microscopy observations and linear dichroism measurements, indicates that the ternary complex remains intact in the presence of DinI, and that the affinity to the RecA filament decreases in the order LexA, DinI and UmuD. DinI is thus suited to modulating UmuD processing so as to limit SOS mutagenesis.

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.

Opposite effects of microtubule-stabilizing and microtubule-destabilizing drugs on biogenesis of mitochondria in mammalian cells.

Distribution of mitochondria as well as other intracellular organelles in mammalian cells is regulated by interphase microtubules. Here, we demonstrate a role of microtubules in the mitochondrial biogenesis using various microtubule-active drugs and human osteosarcoma cell line 143B cells and rat liver-derived RL-34 cells. Depolymerization of microtubules by nocodazole or colchicine, as well as 2-methoxyestradiol, a natural estrogen metabolite, arrested asynchronously cultured cells in G(2)/M phase of cell cycle and at the same time inhibited the mitochondrial mass increase and mtDNA replication. These drugs also inhibited the mitochondrial mass increase in the cells that were synchronized in cell cycle, which should occur during G(1) to G(2) phase progression in normal conditions. However, stabilization of microtubules by taxol did not affect the proliferation of mitochondria during the cell cycle, yet a prolonged incubation of cells with taxol induced an abnormal accumulation of mitochondria in cells arrested in G(2)/M phase of cell cycle. Taxol-induced accumulation of mitochondria was not only demonstrated by mitochondria-specific fluorescent dyes but also evidenced by the examination of cells transfected with yellow fluorescent protein fused with mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (pEYFP) and by enhanced mtDNA replication. Two subpopulations of mitochondria were detected in taxol-treated cells: mitochondria with high Delta(psi)(m), detectable either by Mito Tracker Red CMXRos or by Green FM, and those with low Delta(psi)(m), detectable only by Green FM. However, taxol-induced increases in the mitochondrial mass and in the level of acetylated (alpha)-tubulin were abrogated by a co-treatment with taxol and nocodazole or taxol and colchicine. These data strongly suggest that interphase microtubules may be essential for the regulation of mitochondrial biogenesis in mammalian cells.

High-resolution freeze-etching replica images of the disk and the plasma membrane surfaces in purified bovine rod outer segments.

Molecular organization of the photoreceptor disk membrane was revealed by the freeze-deep etching replica method using purified and successively rinsed bovine rod outer segment (ROS). Various membrane particles with different shape and sizes were found on cytoplasmic surface (PS face) as well as on both P and E fracture faces, which are presumed to be peripheral membrane proteins such as transducin, phosphodiesterase, guanylate cyclase and so on. Membrane particles seen on PS face were catalogued in size. The histogram on their number and size showed that they were classified at least into two major groups, the group of particles about 50 nm2 in size and the group of particles about 115 nm2 in size. The distribution density of the 115 nm2 particle was 1200 microm(-2) in native state, but it decreased to 125 microm(-2) after washing with hypotonic buffer solution. Namely, the group of the 115 nm2-particle seems to be mainly composed of peripheral membrane proteins. Rinsing with the sucrose free buffer at the final step of the purification procedure enabled us to observe three types of filaments localized in ROS (filaments connecting disk to disk at the margin, filaments connecting disk to the plasma membrane, filaments associated with PS face of disk membrane); and also to find characteristic domains with crystal arrangement of particles on the external surface (ES face) of ROS plasma membrane.

Structural change of bovine retinal cGMP phosphodiesterase by release of its gamma subunit: direct imaging by improved low angle rotary shadowing.

Cyclic GMP phosphodiesterase (PDE), a key enzyme for phototransduction, contains two catalytic subunits, Palpha and Pbeta, and two identical regulatory subunits, Pgammas. Neither the structure of the subunits of PDE nor their changes in structure during PDE regulation have been known. Here, improved low angle rotary shadowing was applied to depict the three-dimensional structure of bovine PDE (Palphabetagammagamma) and its changes by Pgamma release. Palphabetagammagamma and Palphabetagamma were isolated from photoreceptor membranes after treatment with a hydrolysis-resistant GTP analogue, and Palphabeta was prepared from Palphabetagammagamma tryptic digestion. Images of Palphabetagammagamma consisted of two crooked strands. These two strands faced each other to make a ring shape, but this ring structure was bent at the centre line between the two strands. In Palphabetagamma, one of these strands changed its shape toward reducing the central space of the ring structure. This ring appeared to be more bent at the centre line. In Palphabeta, both strands changed their shape such that the ring structure appeared to be a twisted quasi ring shape. These observations suggest that in Palphabetagammagamma each Pgamma is complexed with a catalytic subunit, and that the shapes of Palpha and Pbeta are drastically changed by the Pgamma release. These shape changes are no doubt crucial for various PDE regulations, such as activation of cGMP hydrolysis by Palphabeta, interaction of Palphabeta with GARP2 and a GARP2-like protein and cGMP binding to non-catalytic sites on Palphabeta.

ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C.

The eukaryotic DNA sliding clamp that keeps DNA polymerase engaged at a replication fork, called proliferating cell nuclear antigen (PCNA), is loaded onto the 3' ends of primer DNA through its interaction with a heteropentameric protein complex called replication factor C (RFC). The ATPase activity of RFC is necessary for formation of a functional PCNA clamp. In the present study, the sensitivity of RFC to partial proteolysis is used to show that addition of ATP, ATPgammaS, or ADP induces different structural changes in RFC. Direct observation by electron microscopy reveals that RFC has a closed two-finger structure called the U form in the absence of ATP. This is converted into a more open C form on addition of ATP. In contrast, the structural changes induced by ATPgammaS or ADP are limited. These results suggest that RFC adapts on opened configuration intermediately after ATP hydrolysis. We further observe that PCNA is held between the two fingers of RFC and propose that the RFC structure change we observe during ATP hydrolysis causes the attached PCNA to form its active ring-like clamp on DNA.

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.

Direct imaging of phosphorylation-dependent conformational change and DNA binding of CREB by electron microscopy.

BACKGROUND: The second messenger cAMP stimulates the expression of numerous genes through the PKA-dependent phosphorylation of CREB. The cAMP-regulated transcription factor CREB undergoes conformational change in response to phosphorylation by PKA at Ser 133. The phosphorylation enables interaction between the kinase-inducible domain (KID) of CREB and KIX domain of CREB binding protein (CBP). RESULTS: To understand the activation mechanism of CREB-mediated gene expression, we performed the electron-microscope imaging of the transcription machinery. We improved the metal shadowing techniques to achieve higher resolution to detect phosphorylation-induced conformation change of the protein. Homodimer formation of CREB and the complex formation of phosphorylated CREB with CBP were observed under the electron microscope. The binding of the CREB dimer to CREs on the somatostatin and tyrosine hydroxylase promoters were also visualized directly and stereoscopically. CONCLUSIONS: Greatly improved resolution achieved by our modified metal shadowing techniques makes it possible to visualize that the shape of CREB homodimer was changed in phosphorylation-dependent manner and that the promoter DNA strands containing CREs appeared to be bent and twisted slightly by the holding in the crevice of the CREB homodimer. This method may be applicable to visualize transcriptional activation process of nuclear receptors or general transcription machinery.

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. I. Identification of the kinase and its role in the turnoff of phosphodiesterase in vitro.

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H. , Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024-15029), we showed that Pgamma is phosphorylated by a previously unknown kinase (Pgamma kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pgamma loses its ability to interact with GTP/Talpha, but gains a 10-15 times higher ability to inhibit GTP/Talpha-activated PDE than that of nonphosphorylated Pgamma. Thus, we propose that the Pgamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Talpha-activated PDE. Here we demonstrate that all known Pgammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pgamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pgamma indicates that all known Pgammas contain a P-X-T-P-R sequence and that this sequence is required for the Pgamma phosphorylation by Pgamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pgamma kinase activity exactly coelutes with Cdk5 and p35. The Pgamma kinase activity ( approximately 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pgamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pgamma in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pgamma complexed with GTP/Talpha in mature vertebrate retinal photoreceptors.

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo.

Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known Pgammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pgamma phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.

Cryptic dimer interface and domain organization of the extracellular region of metabotropic glutamate receptor subtype 1.

Previously, we produced the whole extracellular region of metabotropic glutamate receptor subtype 1 (mGluR1) in a soluble form. The soluble receptor retained a ligand affinity comparable with that of the full-length membrane-bound receptor and formed a disulfide-linked dimer. Here, we have identified a cysteine residue responsible for the intermolecular disulfide bond and determined domain organization of the extracellular region of mGluR1. A mutant, C140A, was a monomer under nonreduced conditions by SDS-polyacrylamide gel electrophoresis; however, C140A was eluted at the position similar to that of mGluR113, the wild type soluble receptor, by size exclusion column chromatography. Furthermore, C140A bound a ligand, [(3)H]quisqualate, with an affinity similar to that obtained by mGluR113. Oocytes injected with RNA for full-length mGluR1 containing C140A mutation showed responses to ligands at magnitudes similar to those with wild type full-length RNA. Thus, elimination of the disulfide linkage did not perturb the dimer formation and ligand signaling, suggesting that cryptic dimer interface(s) possibly exist in mGluR1. Limited proteolysis of the whole extracellular fragment (residue 33-592) revealed two trypsin-sensitive sites, after the residues Arg(139) and Arg(521). A 15-kDa NH(2)-terminal proteolytic fragment (residue 33-139) was associated with the downstream part after the digestion. Arg(521) was located before a cysteine-rich stretch preceding the transmembrane region. A new shorter soluble receptor (residue 33-522) lacking the cysteine-rich region was designed based on the protease-sensitive boundary. The purified receptor protein gave a K(d) value of 58.1 +/- 0.84 nm, which is compatible to a reported value of the full-length receptor. The B(max) value was 7.06 +/- 0. 82 nmol/mg of protein. These results indicated that the ligand-binding specificity of mGluR1 is confined to the NH(2)-terminal 490-amino acid region of the mature protein.

Functional aspects of megamitochondria isolated from hydrazine- and ethanol-treated rat livers.

It is essential to analyze functions of megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.

Inhibitor peptide SNP-1 binds to a soluble form of BST-1/CD157 at a 2:2 stoichiometry.

Recently we have identified a 15-mer peptide, SNP-1, by a random phage library that can bind to bone marrow stromal cell antigen-1 (BST-1)/CD157 [Sato, A., Yamamoto, S., Ishihara, K., Hirano, T. & Jingami, H. (1999) Biochem. J. 337, 491-496]. SNP-1 inhibits BST-1 ADP-ribosyl cyclase activity uncompetitively with a Ki value of 180 +/- 40 nM. In this study we analysed biophysically the SNP-1 binding to a soluble form of BST-1 (sBST-1). Equilibrium binding data of wild-type SNP-1 from surface plasmon resonance studies gave a Kd value of 500 +/- 35 nM. Titration calorimetry analysis showed that the binding reaction is exothermic at 20 degrees C. The values of Kd = 211 nM, enthalpy change, DeltaH = -18.68 kcal.mol-1, and saturated molar ratio of bound SNP-1 per sBST-1, N = 0.8 mol.mol-1 were obtained. On the basis of the molecular masses of SNP-1 and sBST-1 calculated by analytical ultracentrifugation, the stoichiometry of the binding was determined to be 2 : 2. Electron microscopy also revealed the dimer form of sBST-1. To delineate the core residue of SNP-1 responsible for binding, each amino acid residue has been replaced by alanine. A region from amino acid residues 7-12 appeared to be critical for the SNP-1 binding to sBST-1. The substitution of the first residue, His, to Ala led to a reduction in binding, suggesting that the N-terminal residue is also crucial.

Initiating ocular proteomics for cataloging bovine retinal proteins: microanalytical techniques permit the identification of proteins derived from a novel photoreceptor preparation.

Though some mechanisms of photoreception have been well characterized, others remain obscure. Presumably, most, if not all, of the major players in photoreceptor-specific functions are present in large amounts in the photoreceptor layer, and a catalog of these proteins will prove a useful tool for vision researchers. As a first step toward a complete catalog of photoreceptor cells, we have developed a novel method for isolating the photoreceptor cell monolayer from bovine retina. Electron microscopic studies of both the photoreceptor layer and the residual retina from which the photoreceptor layer had been removed, indicate that the preparation contains the photoreceptor outer segments and the majority of the inner segments. Proteins were extracted from the isolated photoreceptor cell layer as well as the rest of the retina with isoelectric focusing lysis buffer, and the protein components were separated by two-dimensional gel electrophoresis. The obtained protein maps reveal several classes of proteins that appear to be expressed more abundantly or specifically in the photoreceptor layer than in the rest of the retina. Four of these protein spots were excised and in-gel digested with trypsin, and the digests were extracted with solvent. The mixture of peptides digested from each protein was analyzed by high performance liquid chromatography interfaced with electrospray ionization tandem quadrupole mass spectrometry or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Some of the peptides were isolated and their sequences were determined by gas phase Edman degradation. RNA transcripts extracted from the photoreceptor layer or the whole retina were subjected to Northern blot analysis as well as to reverse transcriptase-polymerase chain reaction amplification of probes for the successful selection of cDNA clones. These data permit both the identification of virtually any protein detectable on a two-dimensional gel, and also enable the corresponding cDNA clone to be selected. We have validated this approach by identifying aspartate aminotransferase and creatine kinase from the populations of abundant photoreceptor layer proteins. Both aspartate aminotransferase and creatine kinase are of mitochondrial origin and are thought to play crucial roles in photoreceptor functions by producing glutamate and ATP, respectively. We also identified two photoreceptor layer specific proteins: an acidic and high molecular weight protein, interphotoreceptor retinoid-binding protein, and an acidic and small molecular weight protein, recoverin.The technique presented here will allow vision researchers to discover and identify the proteins that are expressed specifically or abundantly in the photoreceptor cell as well as the proteins that undergo post-translational modification or modulation in expression under a defined biological condition. With the use of this technology, we anticipate that a researcher who knows only the 2-D gel position of a protein of interest can identify the protein, isolate a cDNA clone, and move into molecular genetic studies. Moreover, this streamlined technology will enable one to assemble a catalog of photoreceptor proteins using a minute amount of materials in a short period of time. We believe that such a catalog will serve as a valuable resource for vision investigators and will accelerate the rate of research progress.

Effects of coenzyme Q10 on changes in the membrane potential and rate of generation of reactive oxygen species in hydrazine- and chloramphenicol-treated rat liver mitochondria.

Effects of CoQ10 and cycloheximide (CHX) on hydrazine- and chloramphenicol (CP)-induced morphological and some functional changes of mitochondria using cultured rat hepatocytes and effects on the process of recovery from CP intoxication using mouse liver were examined. Results obtained are summarized as follows: (1) The formation of megamitochondria induced in the hepatocytes cultured for 22 h in the presence of 2 mM hydrazine or CP (300 microgram/ml) was suppressed by pretreatment of hepatocytes with CoQ10 (1 microM) or CHX (0.5 microgram/ml). This was proved by electron microscopic analysis of mitochondria. (2) Treatment of hepatocytes with hydrazine for 48 h or longer caused decreases in the membrane potential of mitochondria, which were suppressed by CoQ10. (3) Treatment of hepatocytes with hydrazine for 22 h or longer caused remarkable increases in intracellular levels of reactive oxygen species in hepatocytes, which were suppressed by CoQ10. (4) The process of recovery from the CP-induced changes of mitochondria in mouse liver was accelerated by CoQ10 and CHX.

Detailed localization of photoreceptor guanylate cyclase activating protein-1 and -2 in mammalian retinas using light and electron microscopy.

Guanylate cyclase activating proteins, GCAP-1 and GCAP-2, have a pivotal role in the activation of guanylate cyclase in phototransduction. Previous studies on the localization of GCAP-1 and GCAP-2 are contradictory. In this study, we tried to avoid possible artifacts accompanied by immunocytochemistry. Immunolabeling of a GCAP was carried out using antibodies pre-adsorbed with a different type of GCAP. In addition, immunolabeling was performed using three different animal species under different fixation and embedding. Electron microscopic immunocytochemistry was also performed to reveal subcellular localization of GCAPs as well as confirming data obtained by light microscopy. All data indicate that anti-GCAP-1 antibody binding sites were found predominantly in cone outer segments, in particular, in disk membrane regions. Sparse labeling was observed in rod outer segments, but the labeling was much lower than that seen in cone outer segments. Less labeling is also found in synaptic regions and inner segments of cones. No labeling was detected in connecting cilia and its cytoplasmic extensions. Such labeling patterns were similar among human, monkey and bovine retinas. The localization of GCAP-1 is consistent with the pattern of a recently reported human cone-specific degeneration. Anti-GCAP-2 antibody binding sites were detected in both inner and outer segments of rods and cones of all three animals although the labeling density was slightly different among species. Cryo-immuno-labeling of GCAP-2 in bovine retinas revealed that labeling sites were more concentrated in rods than those of cones, and that synaptic regions were also labeled. The different localization of GCAPs suggest that roles of GCAP-1 and GCAP-2 may be different.

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.

Periciliary structure of developing rat photoreceptor cells. A deep etch replica and freeze substitution study.

The purpose of this study is to identify the morphological machinery for selective transport of proteins required in the outer segments of the rat photoreceptor cell. As a first step, the three-dimensional architecture of the periciliary region and its developmental changes were examined. Freeze-deep-etching and freeze-substitution methods combined with rapid freezing technique were used. The apical surface of the inner segment was swollen and partially enclosed the base of the connecting cilium in early postnatal stages, so that the basal region of the connecting cilium was inevitably surrounded by a groove. However, a specialized periciliary ridge complex as seen in frog photoreceptor cells has never been observed in rat photoreceptor cells. The cytoplasmic surface of the plasma membrane of the apical inner segment in the vicinity of the connecting cilium was covered with loose fine filaments. However, it was unlikely to be a possible structural candidate for selective transport of membrane proteins. This study also revealed the interior structure of the connecting cilium. Actin filaments in the distal axonem formed a complicated meshwork together with an unknown substance. Since S1 decorated filaments were not detected in the middle region of the connecting cilium, actin filaments at the base of outer segment seem to be independently polymerized locally from G-actin that is transported from the inner segment.