[Oxidative stress and aging arised from mitochondria].
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Adenosine Triphosphate↗
Biomedical subjects
M Tsuda
Publications and source records attributed to M Tsuda.
Action spectrum for the photophobic response of Ciona intestinalis (Ascidieacea, Urochordata) larvae implicates retinal protein.
Ascidian tadpole larvae change swimming behavior during the course of development. The photic behavior of the larvae of Ciona intestinalis was monitored by a computerized cell-tracking system with a time resolution of 0.1 s. Newly hatched larvae swim at an average speed of 1.4 mm/s but show no response to light stimuli. The swimming speed of the larvae became slower (0.4 mm/s) 3 h after hatching and they were induced to swim more rapidly by a sudden decrease in light intensity 4 h after hatching. During the course of development, the maximal speed of swimming behavior increased with time until 8 h after hatching and then plateaued. The action spectrum for the step-down photophobic response of the larvae was determined at around 8 h after hatching and was fitted to Dartnall's nomogram with the absorbance maximum of the pigment located at 505 nm. These results suggest retinal proteins in the ocellus of the larvae are the photoreceptors for the photobehavior.
Animals↗
WT1 nephropathy in a girl with normal karyotype (46,XX)
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Child↗
Inducibility of BDNF gene promoter I detected by calcium-phosphate-mediated DNA transfection is confined to neuronal but not to glial cells.
Although DNA transfection with calcium-phosphate/DNA precipitates for promoter analysis of genes has been previously applied to primary cultures of neuronal cells, it remains uncertain whether the expression of the introduced genes in glial cells, which are also in primary culture, affects the transcriptional signals obtained. Using plasmid DNA containing the brain-derived neurotrophic factor (BDNF) gene promoter I or c-fos promoter, we investigated the optimum conditions for calcium-phosphate-mediated DNA transfection in primary culture of rat cortical neuronal cells. Normalizing the firefly luciferase activity of the experimental reporter gene to the Renilla luciferase activity of the internal control increased experimental reliability. Maximum expression of firefly luciferase activity in the cells stimulated by membrane depolarization required 6-12 hrs of incubation after stimulation. Differences in the ratio of the experimental reporter gene to the internal control did not affect experimental gene expression. Under our optimal conditions, the activation of the BDNF gene promoter I was detected in neuronal but not in glial cells. Calcium-phosphate-mediated DNA transfection should be widely applicable for promoter analysis of inducible genes in neurons.
Animals↗
Molecular cloning and characterization of high-affinity carnitine transporter from rat intestine.
Carnitine is an essential component for mitochondrial beta-oxidation of fatty acid. Using the degenerate primers designed for organic anion transporters and an organic cation transporter, we isolated a novel cDNA encoding a carnitine transporter (CT1) from rat intestine. CT1 encodes a 557-amino-acid protein with 12 putative membrane-spanning domains. When expressed in Xenopus oocytes, CT1 mediated a high-affinity transport of L-carnitine (Km = 25 microM). The replacement of extracellular sodium with Li reduced CT1-mediated L-carnitine uptake to 19.8%. CT1 did not transport typical substrates for either organic anion or organic cation transporters, such as p-aminohippurate and tetraethylammonium. Octanoylcarnitine, acetylcarnitine, and gamma-butyrobetaine showed potent inhibitory effects on CT1-mediated L-carnitine uptake; betaine and d-carnitine showed moderate inhibition. CT1 mRNA was strongly expressed in the testis, colon, kidney, and liver and weakly in the skeletal muscle, placenta, small intestine, and brain. No CT1 expression was detected in the heart, spleen, or lung. The present study provides the molecular basis of carnitine transport in the body.
Amino Acid Sequence↗
Nuclear translocation and increased expression of Bax and disturbance in cell cycle progression without prominent apoptosis induced by hyperthermia.
Effects of hyperthermia at 42.5 degreesC for 6 h on cell survival, cell cycle progression, and the localization and expression levels of Bcl-2 and Bax, as well as the association between Bcl-2 and Bax in human lung cancer cells were investigated. Untreated human lung cancer cells, though immortalized, expressed Bax unlike peripheral lymphocytes with low Bax expression. Bcl-2 was localized only in the cytoplasm in all the cell lines tested, whereas Bax was localized in the cytoplasm and/or nucleus; (1) only in the nucleus in three cell lines, (2) either in the nucleus or the cytoplasm in three cell lines, (3) in both the nucleus and the cytoplasm in one cell line, and (4) only in the cytoplasm in three cell lines. Of 10 cell lines examined, 6 had a low sensitivity to hyperthermia with a viability of 50% or more, and four cell lines had a high sensitivity to hyperthermia with a viability of less than 50% regardless of cell type. In cell lines highly sensitive to hyperthermia, Bax was localized in the nucleus. Hyperthermia increased the cellular level of Bax, but not Bcl-2, and reduced the association between Bcl-2 and Bax expression in PC-10 cells. Although the Bax level increased, hyperthermia induced only mild apoptosis and caused prominent cell cycle disturbance, especially in the S and G2M phases. Thus, hyperthermia at 42.5 degreesC for 6 h had cytostatic effect as well as caused mild apoptosis. Interestingly, during 3 h of hyperthermia, Bax translocated from the cytoplasm to the nucleus, whereas Bcl-2 remained in the cytoplasm. These results raise the possibility that Bax may lose its function as the inducer of apoptosis by translocating into the nucleus or have an unknown role in the nucleus.
Apoptosis↗
A novel photointermediate of octopus rhodopsin activates its G-protein.
The photointermediate of octopus rhodopsin responsible for G-protein activation was examined by a GTPgammaS-binding assay in a reconstituted system with purified rhodopsin and photoreceptor G-protein. When octopus rhodopsin alone was incubated in the dark after illumination, its ability to stimulate GTPgammaS-binding by the G-protein decreased in a time-dependent manner. We associate this decay with the decay of a novel photointermediate, transient acid metarhodopsin, which lies between mesorhodopsin and acid metarhodopsin. Spectroscopic evidence for its existence was suggested by its effects on the turbidity of the vesicles. These results suggest that the transient acid metarhodopsin, not the stable final photoproduct, acid metarhodopsin, activates a G-protein in octopus photoreceptors.
Animals↗
Cancer-associated expression of glycolipid sulfotransferase gene in human renal cell carcinoma cells.
Human renal cell carcinoma (RCC) tissue and a cell line derived therefrom, SMKT-R3, showed markedly increased glycolipid sulfotransferase [cerebroside sulfotransferase (CST); EC 2.8.2.11] activity and accumulated sulfoglycolipids. Recently, we cloned a human CST cDNA from a SMKT-R3 cDNA library (K. Honke et al., J. Biol. Chem., 272: 4864-4868, 1997). In this study, we investigated the expression of the CST gene in seven human RCC lines (SMKT-R1, SMKT-R2, SMKT-R3, SMKT-R4, TOS-1, TOS-2, and ACHN) and their normal counterpart, human renal proximal tubular cells. On Northern blot analysis, a marked increase of CST mRNA was observed in every RCC line, except for ACHN, as compared with normal cells. ACHN cells showed a slightly increased level of CST mRNA. CST activity was correlated with the amount of mRNA. Sulfoglycolipid analysis revealed that expression of lactosylceramide sulfate was correlated with the CST level. Furthermore, we examined the effects of epidermal growth factor (EGF), tetradecanoylphorbol-13-acetate, and genistein, which are known to regulate CST activity in SMKT-R3 cells, on CST-gene expression in various RCC cells. On treatment with EGF, CST mRNA time-dependently increased in accord with its activity in SMKT-R3 cells. Yet, augmentation by EGF was only observed in SMKT-R3. In contrast, a reduction of CST mRNA and activity by tetradecanoylphorbol-13-acetate and genistein was observed in all of the lines examined. Taken together, these findings indicate that in human RCC cells, the CST gene is generally overexpressed via a signaling pathway involving protein kinase-C and tyrosine kinases.
Carcinoma, Renal Cell↗
A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes.
Much attention has focused on the aetiology of oxidative damage in cellular and organismal ageing. Especially toxic are the reactive oxygen byproducts of respiration and other biological processes. A mev-1(kn1) mutant of Caenorhabditis elegans has been found to be hypersensitive to raised oxygen concentrations. Unlike the wild type, its lifespan decreases dramatically as oxygen concentrations are increased from 1 to 60%. Strains bearing this mutation accumulate markers of ageing (such as fluorescent materials and protein carbonyls) faster than the wild type. We show here that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, which is a component of complex II of the mitochondrial electron transport chain. We found that the ability of complex II to catalyse electron transport from succinate to ubiquinone is compromised in mev-1 animals. This may cause an indirect increase in superoxide levels, which in turn leads to oxygen hypersensitivity and premature ageing. Our results indicate that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress.
Aging↗
Functional replacement of OprJ by OprM in the MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa.
For characterization of the MexCD-OprJ efflux system of Pseudomonas aeruginosa involved in resistance to fluoroquinolones and the fourth-generation cephems, we constructed mexC, mexD or oprJ mutants from the nfxB-type PAO strains by insertion mutagenesis. The gene products in the resultant mutants were examined by immunoblot assay using murine and rabbit antibodies developed against purified protein and synthetic oligopeptides. Susceptibility of the mexC (MexC- MexD- OprJ-) and mexD (MexC+ MexD- OprJ-) mutants to fluoroquinolone and the fourth-generation cephems was comparable to that of the wild-type strain PAO1. However, the oprJ mutant (MexC+ MexD+ OprJ-) was still less susceptible than PAO1, since a MexCD-OprM chimera system, which generated from a functional assist of the constitutively expressed OprM, can function in the efflux of the antimicrobial agents in the oprJ mutant. In fact, transformation of the oprJ mutant with an OprM-expression plasmid decreased the former's susceptibility to the levels exhibited by the nfxB mutant without affecting the substrate specificity of MexCD-OprJ.
Animals↗
Cloning and sequencing of a novel Na+/H+ antiporter gene from Pseudomonas aeruginosa.
We cloned a gene for Na+/H+ antiporter from chromosomal DNA of Pseudomonas aeruginosa. Introduction of the gene into host Escherichia coli mutant cells lacking all of the major Na+/H+ antiporters enabled the cells to grow in the presence of 0.2 M NaCl, although the original host cells could not. Membrane vesicles prepared from cells of the transformant possessing the cloned gene showed Na+/H+ antiport activity. As a result of DNA sequencing, we found one open reading frame (nhaP). The deduced amino acid sequence suggests that the Na+/H+ antiporter (NhaP) of P. aeruginosa consists of 424 amino acid residues with molecular mass of 45486 Da, and hydropathy analysis suggested the presence of 12 putative transmembrane domains. We found no bacterial Na+/H+ antiporter which showed significant sequence similarity with the NhaP in the protein sequence database. The NhaP showed partial sequence similarity with animal Na+/H+ exchangers. Thus, the NhaP of P. aeruginosa is unique among bacterial antiporters.
Amino Acid Sequence↗
Conversion of temperature-sensitive to -resistant gene expression due to mutations in the promoter region of the melibiose operon in Escherichia coli.
The melibiose utilization system of Escherichia coli W3133, a derivative of K12, is nonfunctional between 37 and 42 degreesC. The reason for this temperature sensitivity was thought to be that the melibiose transporter (MelB) of W3133 cells was temperature-sensitive. A mutant W3133-2 has been isolated as a temperature-resistant strain that can utilize melibiose between 37 and 42 degreesC. However, we found that the melibiose transporter of the W3133-2 was still temperature-sensitive. Half-life activities of the melibiose transporter at 37 degreesC (or 40 degreesC) in both E. coli W3133 and W3133-2 were exactly the same. Furthermore, we found that the nucleotide sequence of coding region of the melB structural gene (the second gene of the melibiose operon) of W3133-2 was exactly the same as that of W3133. Activity of alpha-galactosidase (product of the first gene, melA, of the melibiose operon) of W3133 cells grown at 40 degreesC was very low, although that of W3133-2 cells grown at 40 degreesC was high. These observations suggested that expression of the melibiose operon in W3133 is also temperature-sensitive. In fact, we found that the expression in W3133 cells was temperature-sensitive, while that in W3133-2 cells was temperature-resistant, by analyzing mRNA levels using the Northern blot method. Furthermore, we identified mutations in the promoter region of the melibiose operon of W3133-2 that resulted in the elongation of an 18 nucleotide inverted repeat sequence to a 28-nucleotide repeat sequence present immediately upstream of the -35 region. This may stabilize a possible stem structure due to the inverted repeat at 37-42 degreesC.
Base Sequence↗
Identification of multispecific organic anion transporter 2 expressed predominantly in the liver.
In the present study, we demonstrate that NLT (novel liver-specific transport protein) is a multispecific organic anion transporter of the liver. The amino acid sequence of NLT shows 42% identity to that of the renal multispecific organic anion transporter, OAT1. When expressed in Xenopus laevis oocytes, NLT mediated uptake of organic anions, such as salicylate, acetylsalicylate, PGE2, dicarboxylates and p-aminohippurate. [14C]Salicylate uptake via NLT was saturable (Km = 88.8 +/- 23.4 microM) and sodium-independent. Expression of the mRNA of NLT was detected in the liver and kidney (liver >> kidney). We propose that NLT be renamed OAT2.
Animals↗
Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: roles of RHO, FAK, and a SRC family kinase.
SHPS-1 is an approximately 120 kDa glycosylated receptor like protein that contains three immunoglobulin-like domains in its extracellular region as well as four potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites in its cytoplasmic region. Lysophosphatidic acid (LPA) stimulated the rapid tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, a protein tyrosine phosphatase containing SH2 domains in Rat-1 fibroblasts. LAP-induced tyrosine phosphorylation of SHPS-1 was inhibited by Clostridium botulinum C3 exoenzyme (which inactivates RHO) but not by pertussis toxin. The protein kinase C activator phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulated tyrosine phosphorylation of SHPS-1; however, down-regulation of protein kinase C by prolonged exposure of cells to TPA did not affect LAP-induced tyrosine phosphorylation of SHPS-1. LPA-induced tyrosine phosphorylation of SHPS-1 was markedly reduced in either focal adhesion kinase (FAK)-deficient mouse cells or CHO cells overexpressing the tyrosine kinase CSK. Overexpression of a catalytically inactivate SHP-2 markedly inhibited MAP kinase activation in response to low concentrations of LPA in CHO cells, whereas overexpression of a wild-type SHPS-1 did enhance this effect of LPA. Furthermore, MAP kinase activation in response to a low concentration of LPA was inhibited by botulinum C3 exoenzyme. These results indicate that LPA-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2 may be mediated by a RHO-dependent pathway that includes FAK and a SRC family kinase. Thus, in addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, the formation of a complex between SHPS-1 and SHP-2 may, in part, play an important role in the activation of MAP kinase in response to low concentrations of LPA.
ADP Ribose Transferases↗
Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Roles of Fak and Src family kinases.
SHPS-1 is a receptor-like glycoprotein that undergoes tyrosine phosphorylation and binds SHP-2, an Src homology 2 domain containing protein tyrosine phosphatase, in response to various mitogens. Cell adhesion to extracellular matrix proteins such as fibronectin and laminin also induced the tyrosine phosphorylation of SHPS-1 and its association with SHP-2. These responses were markedly reduced in cells overexpressing the Csk kinase or in cells that lack focal adhesion kinase or the Src family kinases Src or Fyn. However, unlike Src, focal adhesion kinase did not catalyze phosphorylation of the cytoplasmic domain of SHPS-1 in vitro. Overexpression of a catalytically inactive SHP-2 markedly inhibited activation of mitogen-activated protein (MAP) kinase in response to fibronectin stimulation without affecting the extent of tyrosine phosphorylation of focal adhesion kinase or its interaction with the docking protein Grb2. Overexpression of wild-type SHPS-1 did not enhance fibronectin-induced activation of MAP kinase. These results indicate that the binding of integrins to the extracellular matrix induces tyrosine phosphorylation of SHPS-1 and its association with SHP-2, and that such phosphorylation of SHPS-1 requires both focal adhesion kinase and an Src family kinase. In addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, SHP-2 may play an important role, partly through its interaction with SHPS-1, in the activation of MAP kinase in response to the engagement of integrins by the extracellular matrix.
Animals↗
NMDA receptor antagonists potently suppress the spontaneous withdrawal signs induced by discontinuation of long-term diazepam treatment in Fischer 344 rats.
The present study investigated the effects of the NMDA receptor antagonists dizocilpine (MK-801) and ifenprodil on the appearance of diazepam withdrawal signs caused by discontinuation of long-term diazepam treatment using a drug-admixed food (DAF) method in Fischer 344 rats. The total withdrawal score was significantly decreased by after-withdrawal treatment with dizocilpine or ifenprodil. Dizocilpine, in particular, markedly suppressed the motor withdrawal signs and body weight loss, while ifenprodil suppressed the motor and emotional withdrawal signs. Furthermore, the decrease in the food intake during withdrawal (anorexia) was significantly reduced by dizocilpine, but not by ifenprodil. These behavioral results indicated that the activation of NMDA receptors during withdrawal may play an important role in the appearance of withdrawal signs (in particular motor withdrawal signs) caused by discontinuation of chronic diazepam treatment, and that inhibitory agents for NMDA receptors may be effective in alleviation of the appearance of benzodiazepine withdrawal signs.
Administration, Oral↗
Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation.
SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.
Amino Acid Sequence↗
A novel rhodopsin kinase in octopus photoreceptor possesses a pleckstrin homology domain and is activated by G protein betagamma-subunits.
G protein-coupled receptor kinases (GRKs) play an important role in stimulus-dependent receptor phosphorylation and desensitization of the receptors. Mammalian rhodopsin kinase (RK) and beta-adrenergic receptor kinase (betaARK) are the most studied members among known GRKs. In this work, we purified RK from octopus photoreceptors for the first time from invertebrate tissues. The molecular mass of the purified enzyme was 80 kDa as estimated by SDS-polyacrylamide gel electrophoresis, and this was 17 kDa larger than that of the vertebrate enzymes. Unlike vertebrate RK, octopus RK (ORK) was directly activated by betagamma-subunits of a photoreceptor G protein. We examined the effects of various known activators and inhibitors of GRKs on the activity of the purified ORK and found that their effects were different from those on either bovine RK or betaARK. To analyze the primary structure of the enzyme, we cloned the cDNA encoding ORK from an octopus retinal cDNA library. The deduced amino acid sequence of the cDNA was highly homologous to betaARK over the entire molecule, including a pleckstrin homology domain located in the C-terminal region, and homology to RK was significantly lower. Furthermore, Western blot analysis of various octopus tissues with an antibody against the purified ORK showed that ORK is expressed solely in the retina, which confirmed the identity of the enzyme as rhodopsin kinase. Thus, ORK appears to represent a unique subgroup in the GRK family, which is distinguished from vertebrate RK.
Amino Acid Sequence↗