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Biomedical subjects

Takashi Watanabe

Publications and source records attributed to Takashi Watanabe.

At least 55 records · Page 3Linked to original sources
PubMed2005

Engineering functional changes in Escherichia coli endonuclease III based on phylogenetic and structural analyses.

Escherichia coli endonuclease III (EcoNth) plays an important cellular role by removing premutagenic pyrimidine damages produced by reactive oxygen species. EcoNth is a bifunctional enzyme that has DNA glycosylase and apurinic/apyrimidinic lyase activities. Using a phylogeny of natural sequences, we selected to study EcoNth serine 39, aspartate 44, and arginine 184, which are presumed to be in the vicinity of the damaged base in the glycosylase-substrate complex. These three amino acids are highly conserved among Nth orthologs, although not among homologous glycosylases, such as MutY, that have different base specificities and no lyase activity. To examine the role of these amino acids in catalysis, we constructed three mutants of EcoNth, in which Ser39 was replaced with leucine (S39L), Asp44 was replaced with valine (D44V), and Arg184 was replaced with alanine (R184A), which are the corresponding residues in EcoMutY. We showed that EcoNth S39L does not have significant glycosylase activity for oxidized pyrimidines, although it maintained AP lyase activity. In contrast, EcoNth D44V retained glycosylase activity against oxidized pyrimidines, but the apparent rate constant for the lyase activity of EcoNth D44V was significantly lower than that of EcoNth, indicating that Asp44 in EcoNth is required for beta-elimination. Finally, EcoNth R184A maintained lyase activity but exhibited glycosylase specificity different from that of EcoNth. The functional consequences of each of these three substitutions can be rationalized in the context of high resolution protein structures. Thus phylogeny-based scanning mutagenesis has allowed us to identify novel roles for amino acids in the substrate binding pocket of EcoNth in base recognition and/or catalysis.

Arginine↗
PubMed2005

Essential role of protein kinase B gamma (PKB gamma/Akt3) in postnatal brain development but not in glucose homeostasis.

Protein kinase B is implicated in many crucial cellular processes, such as metabolism, apoptosis and cell proliferation. In contrast to Pkb(alpha) and Pkb(beta)-deficient mice, Pkb(gamma)(-/-) mice are viable, show no growth retardation and display normal glucose metabolism. However, in adult Pkb(gamma)mutant mice, brain size and weight are dramatically reduced by about 25%. In vivo magnetic resonance imaging confirmed the reduction of Pkb(gamma)(-/-) brain volumes with a proportionally smaller ventricular system. Examination of the major brain structures revealed no anatomical malformations except for a pronounced thinning of white matter fibre connections in the corpus callosum. The reduction in brain weight of Pkb(gamma)(-/-) mice is caused, at least partially, by a significant reduction in both cell size and cell number. Our results provide novel insights into the physiological role of Pkb(gamma) and suggest a crucial role in postnatal brain development.

Animals↗
PubMed2005

Mutation analysis of the human 5-lipoxygenase C-terminus: support for a stabilizing C-terminal loop.

Lipoxygenases contain prosthetic iron, in human 5-lipoxygenase (5LO) the C-terminal isoleucine carboxylate constitutes one of five identified ligands. ATP is one of several factors determining 5LO activity. We compared properties of a series of 5LO C-terminal deletion mutants (one to six amino acid residues deleted). All mutants were enzymatically inactive (expected due to loss of iron), but expression yield (in E. coli) and affinity to ATP-agarose was markedly different. Deletion of up to four C-terminal residues was compatible with good expression and retained affinity to the ATP-column, as for wild-type 5LO. However when also the fifth residue was deleted (Asn-669) expression yield decreased and the affinity to ATP was markedly diminished. This was interpreted as a result of deranged structure and stability, due to loss of a hydrogen bond between Asn-669 and His-399. Mutagenesis of these residues supported this conclusion. In the structure of soybean lipoxygenase-1, a C-terminal loop was pointed out as important for correct orientation of the C-terminus. Accordingly, a hydrogen bond appears to stabilize such a C-terminal loop also in 5LO.

Adenosine Triphosphate↗
PubMed2005

IQGAP1: a key regulator of adhesion and migration.

The dynamic rearrangement of cell-cell adhesion is one of the major physiological events in tissue development and tumor metastasis. Polarized cell migration, another key event, is a tightly regulated process that occurs during tissue development, chemotaxis and wound healing. Rho-family small GTPases, especially Rac1 and Cdc42, play pivotal roles in these processes through one of their effectors, IQGAP1. Recent studies reveal that IQGAP1 regulates cadherin-mediated cell-cell adhesion both positively and negatively. It captures and stabilizes microtubules through the microtubule-binding protein CLIP-170 near the cell cortex, leading to establishment of polarized cell morphology and directional cell migration. Furthermore, Rac1 and Cdc42 link the adenomatous polyposis coli (APC) protein to actin filaments through IQGAP1 at the leading edge and thereby regulate polarization and directional migration.

Actins↗
PubMed2005

ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic beta cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy.

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic beta cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic beta cells.

Animals↗
PubMed2005

Spatial resolution of calpain-catalyzed proteolysis in focal cerebral ischemia.

Transient forebrain ischemia induces calpain-mediated degradation of the neuronal cytoskeleton, alpha-fodrin, and this results in ischemic neuronal death. In this study, we investigated the spatial distribution and temporal changes of calpain-catalyzed alpha-fodrin proteolysis in focal cerebral ischemia and examined the effects of a calpain inhibitor. Ischemia was induced in gerbils by 3-h middle cerebral artery occlusion followed by reperfusion. Animals were divided into four groups: a sham-operated group, an ischemic group, a vehicle-treated group, and a calpain inhibitor-treated group. Intravenous injections of vehicle or calpain inhibitor I were administered 30 min before ischemia. Infarct volumes were measured 1 day after reperfusion and the spatial distribution of calpain-catalyzed alpha-fodrin proteolysis was investigated by immunohistochemistry 15 min, 1 h, 4 h, and 1 day after reperfusion. Infarct volume (mean +/- SD) in the ischemic group and the vehicle-treated group was 204.6 +/- 19.1 mm3 and 212.4 +/- 16.3 mm3, respectively, and the calpain inhibitor I reduced the infarct volume [149.4 +/- 25.2 mm3 (P < 0.05)]. Immunoblot analysis demonstrated that calpain inhibitor reduced proteolysis. Ischemia induced fodrin proteolysis in the ischemic core and the peri-infarct zone within 15 min after reperfusion, with proteolysis developing quickly in the ischemic core and more slowly in the peri-infarct zone. Proteolysis preceded neuronal death in the peri-infarct zone. Calpain inhibitor I ameliorated neuronal death in the peri-infarct zone but not in the ischemic core. Thus, calpain plays a pivotal role on focal ischemia as well as in global ischemia.

Animals↗
PubMed2005

Transdifferentiation of the retinal pigment epithelia to the neural retina by transfer of the Pax6 transcriptional factor.

The Pax6 gene plays an important role in eye morphogenesis throughout the animal kingdom. The Pax6 gene and its homologue could form ectopic eyes by targeted expression in Drosophila and Xenopus. Thus, this gene is a master gene for the eye morphogenesis at least in these animals. In the early development of the vertebrate eye, Pax6 is required for the instruction of multipotential progenitor cells of the neural retina (NR). Primitive retinal pigment epithelial (RPE) cells are able to switch their phenotype and differentiate into NR under exogenous intervention, including treatment with fibroblast growth factors (FGFs), and surgical removal of endogenous NR. However, the molecular basis of phenotypic switching is still controversial. Here, we show that Pax6 alone is sufficient to induce transdifferentiation of ectopic NR from RPE cells without addition of FGFs or surgical manipulation. Pax6-mediated transdifferentiation can be induced even at later stages of development. Both in vivo and in vitro studies show that the Pax6 lies downstream of FGF signaling, highlighting the central roles of Pax6 in NR transdifferentiation. Our results provide an evidence of retinogenic potential of nearly mature RPE and a cue for new therapeutic approaches to regenerate functional NR in patients with a visual loss.

Animals↗
PubMed2005

Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isoenzyme from Pleurotus ostreatus without redox mediators.

VPs (versatile peroxidases) sharing the functions of LiP (lignin peroxidase) and MnP (manganese peroxidase) have been described in basidiomycetous fungi Pleurotus and Bjerkandera. Despite the importance of this enzyme in polymer degradation, its reactivity with polymeric substrates remains poorly understood. In the present study, we first report that, unlike LiP, VP from Pleurotus ostreatus directly oxidized two polymeric substrates, bovine pancreatic RNase and Poly R-478, through a long-range electron pathway without redox mediators. P. ostreatus produces several MnP isoenzymes, including the multifunctional enzyme MnP2 (VP) and a typical MnP isoenzyme MnP3. MnP2 (VP) depolymerized a polymeric azo dye, Poly R-478, to complete its catalytic cycle. Reduction of the oxidized intermediates of MnP2 (VP) to its resting state was also observed for RNase. RNase inhibited the oxidation of VA (veratryl alcohol) in a competitive manner. Blocking of the exposed tryptophan by N-bromosuccinimide inhibited the oxidation of RNase and VA by MnP2 (VP), but its Mn2+-oxidizing activity was retained, suggesting that Trp-170 exposed on an enzyme surface is a substrate-binding site both for VA and the polymeric substrates. The direct oxidation of RNase and Poly R by MnP2 (VP) is in sharp contrast with redox mediator-dependent oxidation of these polymers by LiP from Phanerochaete chrysosporium. Molecular modelling of MnP2 (VP) revealed that the differences in the dependence on redox mediators in polymer oxidation by MnP2 (VP) and LiP were explained by the anionic microenvironment surrounding the exposed tryptophan.

Anthraquinones↗
PubMed2005

Examining SLV-323, a novel NK1 receptor antagonist, in a chronic psychosocial stress model for depression.

RATIONALE: Substance P antagonists have been proposed as candidates for a new class of antidepressant compounds. OBJECTIVES: We examined the effects of SLV-323, a novel neurokinin 1 receptor (NK1R) antagonist, in the chronic psychosocial stress paradigm of adult male tree shrews. METHODS: Animals were subjected to a 7 day period of psychosocial stress before being treated daily with SLV-323 (20 mg kg(-1) day(-1)). The psychosocial stress continued throughout the treatment period of 28 days. Brain metabolite concentrations were determined in vivo by proton magnetic resonance spectroscopy. Norepinephrine excretion was monitored from daily urine samples, and serum testosterone concentrations were measured at the end of the experiment. All animals were videotaped daily to analyze scent-marking behavior and locomotor activity. Cell proliferation in the dentate gyrus and hippocampal volume were measured postmortem. RESULTS: Stress significantly decreased cerebral concentrations of N-acetyl-aspartate, total creatine, and choline-containing compounds in vivo and resulted in an increase of urinary norepinephrine and decrease of serum testosterone concentrations. Moreover, stressed animals displayed decreased scent-marking behavior and locomotor activity. The proliferation rate of the granule precursor cells in the dentate gyrus was reduced, and hippocampal volume was mildly decreased. The stress-induced alterations in the central nervous system were partially prevented by concomitant administration of SLV-323, while drug treatment had only a minor effect on the stress-induced behavioral changes. CONCLUSIONS: The novel NK1R antagonist SLV-323 has certain antidepressant-like effects in a valid animal model of depression.

Animals↗
PubMed2005

Mn(2+) is dispensable for the production of active MnP2 by Pleurotus ostreatus.

The regulation mechanism for expression of versatile peroxidase MnP2 by the basidiomycete fungus Pleurotus ostreatus was examined using chemically defined synthetic media. Expression of MnP2 was down-regulated at the transcription level by nutrient nitrogen, e.g., NH(4)(+), arginine or urea. As is often the case with other fungal manganese peroxidases, active MnP2 was not detected when Mn(2+) was omitted from the culture, while mnp2 transcription was barely affected by Mn(2+). However, Mn(2+) can be substituted by an MnP2 substrate, Poly R-478, since active MnP2 was detected extracellularly when the compound was added to the culture without Mn(2+). Enzyme stability assays with the purified MnP2 indicated an indispensable requirement for a substrate that can be used to complete the catalytic cycle, and avoid inactivation resulting from an excess H(2)O(2). This report is the first of the Mn(2+)-independent production of an active versatile peroxidase by P. ostreatus.

Catalysis↗
PubMed2005

The Pax6 isoform bearing an alternative spliced exon promotes the development of the neural retinal structure.

The vertebrate retina has an area where visual cells are closely packed for proper vision that is known as a fovea, an area centralis or a visual streak. The molecular mechanism that regulates the formation of these structures and visual cell gradients is unknown. The transcription factor Pax6 is a master regulator of eye development. A Pax6 isoform that contains an exon 5a-encoded 14 amino acid insertion in its paired domain, Pax6(+5a), has different DNA-binding properties compared with the Pax6(-5a) isoform. Little is known about the functional significance of Pax6(+5a). Here, we show that Pax6(+5a) is expressed especially in the retinal portion where visual cells accumulate during eye development and, when overexpressed, induces a remarkable well-differentiated retina-like structure. Pax6(+5a) proteins that bear point mutations that are found in patients with foveal hypoplasia are unable to induce these ectopic retina-like structures. We propose that Pax6(+5a) induces a developmental cascade in the prospective fovea, area centralis or visual streak region that leads to the formation of a retinal architecture bearing densely packed visual cells.

Alternative Splicing↗
PubMed2005

Cell cycle dependence of elicitor-induced signal transduction in tobacco BY-2 cells.

The molecular links between the cell cycle and defense responses in plants are largely unknown. Using synchronized tobacco BY-2 cells, we analyzed the cell cycle dependence of elicitor-induced defense responses. In synchronized cultured apoaequorin-expressing cells, the increase in cytosolic free Ca2+ induced by a proteinaceous elicitor, cryptogein, was greatly suppressed during the G2 and M phases in comparison with G1 or S phases. Treatment with cryptogein during the G1 or S phases also induced biphasic (rapid/transient and slow/prolonged) responses in activation of mitogen-activated protein kinases (MAPKs) and production of reactive oxygen species (ROS). In contrast, elicitor treatment during the G2 or M phases induced only a rapid and transient phase of MAPK activation and ROS production. Their slow and prolonged phases as well as expression of defense-related genes, cell cycle arrest and cell death were induced only after the cell cycle progressed to the G1 phase; removal of the elicitor before the start of the G1 phase inhibited these responses. These results suggest that although cryptogein recognition occurred at all phases of the cell cycle, the recognition during the S or G1 phases, but not at the G2 or M phases, induces the prolonged activation of MAPKs and the prolonged production of ROS, followed by cell cycle arrest, accumulation of defense-related gene transcripts and cell death. Elicitor signal transduction depends on the cell cycle and is regulated differently at each phase.

Aequorin↗
PubMed2005

In vivo 3D MRI of insect brain: cerebral development during metamorphosis of Manduca sexta.

High-resolution 3D MRI of male pupae of Manduca sexta was performed at 2.35 T in order to evaluate its potential for an in vivo characterization of insect brain during metamorphosis. T1-weighted 3D FLASH (TR/TE = 20/7.8 ms, 25 degrees flip angle) and T2-weighted 3D fast SE MRI data sets (TR/TEeff = 3000/100 ms) were acquired at different developmental stages with an isotropic resolution of 100 microm. Both T1- and T2-weighted 3D MRI allowed for the identification of cerebral structures such as the antennal nerve, antennal and optical lobe, and central brain. Pronounced developmental alterations of the morphology were observed during metamorphosis. The results demonstrate the feasibility of 3D MRI at nanoliter resolution to identify major brain systems of M. sexta and respective changes during pupal development from caterpillar to sphinx moth. Together with the use of suitable contrast agents, this approach may provide new ways for studying the axonal connectivity and neural function of the developing insect brain.

Animals↗
PubMed2005

Functional role of a novel ternary complex comprising SRF and CREB in expression of Krox-20 in early embryos of Xenopus laevis.

Krox-20, originally identified as a member of "immediate-early" genes, plays a crucial role in the formation of two specific segments in the hindbrain during early development of the vertebrate nervous system. Here we cloned a genomic sequence of Xenopus Krox-20 (XKrox-20) and studied functions of a promoter element in the flanking sequence and associated transcription factors, which function in early Xenopus embryos. Using the luciferase reporter assay system, we showed that the 5' flanking sequence was sufficient to induce luciferase activities when the reporter construct was injected into embryos at the eight-cell stage. Deletion and mutagenesis analyses of the 5' flanking sequence revealed a minimal promoter element that included two known subelements, a CArG-box and cAMP response element (CRE) within a stretch of 22 bp nucleotide sequence (-72 to -51 from the transcription initiation site), both of which were essential for the promoter activity. The gel mobility shift assay indicated that these two subelements bound to some components in whole cell extracts prepared from stage 20 Xenopus embryos. Antibody supershift and competition experiments revealed that these components in cell extracts were serum response factor (SRF) and a member of CRE binding protein (CREB) family proteins that bound the CArG-box and CRE, respectively. They appeared to assemble on the minimal promoter element to produce a novel ternary complex. When we injected mRNA of a dominant-negative version of Xenopus SRF (XSRFDeltaC) into animal pole blastomeres at the eight-cell stage, expression of XKrox-20 in the nervous system as well as the minimal promoter activity was strongly suppressed. Suppression by XSRFDeltaC was counteracted by coexpressed wild-type XSRF. These results indicate that XSRF functions as an endogenous activator of XKrox-20 by forming a ternary complex with CREB on the minimal promoter element.

Animals↗
PubMed2005

The cyclooxygenase inhibitor indomethacin modulates gene expression and represses the extracellular matrix protein laminin gamma1 in human glioblastoma cells.

The induction of cyclooxygenase-2 (COX-2) expression is associated with more aggressive gliomas and poor survival. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and have antitumorigenic properties. In this report, our initial aim was to determine if indomethacin would alter gene expression as measured by suppression subtractive hybridization (SSH). Three up-regulated and four down-regulated genes by indomethacin treatment were identified. Laminin gamma1, an extracellular matrix molecule, was the most significantly repressed gene. The repression of laminin gamma1 by indomethacin was confirmed by Northern and Western blot analyses and occurred in a concentration- and time-dependent manner at the protein level. Among several NSAIDs tested, only sulindac sulfide and indomethacin suppressed laminin gamma1 protein expression, and this repression was observed in both COX-expressing and -deficient cell lines, suggesting that laminin gamma1 repression by COX inhibitors was independent of COX. Indomethacin, at a concentration that represses laminin gamma1, inhibited glioblastoma cell invasion that was partially restored with additional human laminin protein containing gamma1 chain. The repression of laminin gamma1 by NSAIDs may be related to attenuation of invasion of brain tumors. These findings are important in understanding the chemopreventive activity of some NSAIDs and could be relevant for designing therapeutic strategies against glioblastoma.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Successful removal of an infected pacemaker lead using cardiopulmonary bypass in an 89-year-old patient.

We report the case of an 89-year-old patient suffering from endocarditis with septicemia caused by a growth on a pacemaker lead. The entire pacemaker system was successfully removed using cardiopulmonary bypass. Although the patient was an octogenarian in poor condition with a systemic infection, an aggressive operation with careful perioperative management gave a good clinical result. As far as we know, this is the oldest patient in whom a pacemaker system has been removed using cardiopulmonary bypass.

Aged, 80 and over↗
PubMed2005

Learning curve of coronary surgery by a cardiac surgeon in Japan with the use of cumulative sum analysis.

OBJECTIVE: Case-volume has been believed to be of paramount importance in becoming a good cardiac surgeon. However, for a training cardiac surgeon there is no evidence regarding the specific number of necessary cases to attain the medico-legally acceptable level. We attempted to observe the learning curve in performing coronary surgery with the use of the cumulative sum (CUSUM) method to provide some evidence. METHODS: From April 2000 to March 2004, a cardiac surgeon, being board-certified but not being on the attending staff, performed 50 coronary artery bypass grafting (CABG) under supervision of three different chief attending surgeons at three different hospitals. His learning curve was analyzed with the use of CUSUM. Mortality and morbidity and the average time of crossclamp and operation at each hospital were examined. Also, the learning curve of 100 left internal mammary artery (LIMA) harvestings were analyzed via CUSUM in terms of harvesting time and injury rate. RESULTS: The CUSUM curve tended to come closer to the alert line (0.80 confidence) until the 23rd case, but never reached the alert line thereafter until the 50th case. The CUSUM curve never transgressed the alarm line (0.95 confidence) throughout 50 cases. The CUSUM curve of LIMA harvesting approached the alert line in the 38th case and the 59th case, but thereafter never reached the alert line and remained below the reassurance line from the 73rd case. CONCLUSION: Tentatively, it is inferred that approximately 23 cases of CABG may be sufficient to allow for independent practice and that 73 cases of LIMA harvesting are sufficient to allow independent practice.

Adult↗