Effect of site-specific mutagenesis of tyrosine-55, methionine-110 and histidine-217 in porcine kidney D-amino acid oxidase on its catalytic function

AbstractTo assess the contributions of Tyr-55, Met-110 and His-217 in porcine kidney D-amino acid oxidase (EC 1.4.3.3, DAO) to its catalytic function, we constructed three mutant cDNAs coding for the enzymes possessing Phe-55, Leu-110 and Leu-217 by site-specific mutagenesis. The mutant and wild type cDNAs could be expressed in vitro with similar efficiency. The three mutant enzymes thus synthesized showed catalytic activities comparable to that of the wild type oxidase. It is concluded that Tyr-55, Met-110 and His-217 are not directly involved in the catalytic function

トクシマ ダイガク シラギクカイ ノ カツドウ ト コンゴ ノ ホウコウセイ

Shiragiku-kai, established in 1967, is a nonprofit organization whose members are individually registered for the willed body donation program at Tokushima University. Main activities of Shiragiku-kai include returning the remains after studies are complete, holding annual memorial services of donors, and organizing general and district assembly meetings. Currently, twelve clinical departments are conducting surgical training and clinical research in the Clinical Anatomy Laboratory utilizing donations. To meet the ongoing need for donations, it is essential to promote greater understanding among the general public and increase the number of registered members for this valuable program

Expression of tyrosine hydroxylase in cerebellar Purkinje cells of ataxic mutant mice:its relation to the onset and/or development of ataxia

This report describes recent studies on tyrosine hydroxylase (TH) expression in Purkinje cells of the cerebellum of ataxic mutant mice. An increased expression of TH in some Purkinje cells has been observed in two allelic groups of mutant mice, tottering and dilute. TH-positive Purkinje cells appeared preceding the onset of ataxia. Northern blot analysis revealed2.1kb of TH mRNA in the mutant cerebella, and the size was identical to that of TH transcripts in other brain regions. However, TH in Purkinje cells did not seem to participate in catecholamine biosynthesis. In vitro studies showed that cultured non-catecholaminergic neurons expressed the TH transcripts following Ca2+ influx. Therefore, abnormal TH expression in the mutant Purkinje cells may indicate neuronal dysfunction caused by misregulation of intracellular Ca2+concentrations

Inactivation of the DNA Repair Genes mutS, mutL or the Anti-Recombination Gene mutS2 Leads to Activation of Vitamin B1 Biosynthesis Genes

Oxidative stress generates harmful reactive oxygen species (ROS) that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B1, which can serve as an ROS scavenger. In addition, disruption of mutS, mutL, or mutS2 resulted in an increased rate of oxidative stress-induced mutagenesis. Co-immunoprecipitation and pull-down experiments revealed previously-unknown interactions of MutS2 with MutS and MutL, indicating that these proteins cooperatively participate in the repair of oxidatively damaged DNA. These results suggested that bacterial cells sense the accumulation of oxidative DNA damage or absence of DNA repair activity, and signal the information to the transcriptional regulation machinery for an ROS-detoxifying system

Determination of ADAMTS13 and Its Clinical Significance for ADAMTS13 Supplementation Therapy to Improve the Survival of Patients with Decompensated Liver Cirrhosis

The liver plays a central role in hemostasis by synthesizing clotting factors, coagulation inhibitors, and fibrinolytic proteins. Liver cirrhosis (LC), therefore, impacts on both primary and secondary hemostatic mechanisms. ADAMTS13 is a metalloproteinase, produced exclusively in hepatic stellate cells, and specifically cleaves unusually large von Willebrand factor multimers (UL-VWFM). Deficiency of ADAMTS13 results in accumulation of UL-VWFM, which induces platelet clumping or thrombi under high shear stress, followed by sinusoidal microcirculatory disturbances and subsequent progression of liver injuries, eventually leading to multiorgan failure. The marked imbalance between decreased ADAMTS13 activity (ADAMTS13 : AC) and increased production of UL-VWFM indicating a high-risk state of platelet microthrombi formation was closely related to functional liver capacity, hepatic encephalopathy, hepatorenal syndrome, and intractable ascites in advanced LC. Some end-stage LC patients with extremely low ADAMTS13 : AC and its IgG inhibitor may reflect conditions similar to thrombotic thrombocytopenic purpura (TTP) or may reflect “subclinical TTP.” Hence, cirrhotic patients with severe to moderate deficiency of ADAMTS13 : AC may be candidates for FFP infusion as a source of ADAMTS13 or for recombinant ADAMTS13 supplementation. Such treatments may improve the survival of patients with decompensated LC

DOI mitigates DNT of EtOH to 5-HT cells

Prenatal ethanol exposure causes the reduction of serotonergic (5-HTergic) neurons in the midbrain raphe nuclei. In the present study, we examined whether an activation of signaling via 5-HT2A and 5-HT2C receptors during the fetal period is able to prevent the reduction of 5-HTergic neurons induced by prenatal ethanol exposure. Pregnant Sprague-Dawley rats were given a liquid diet containing 2.5 to 5.0% (w/v) ethanol on gestational days (GDs) 10 to 20 (Et). As a pair-fed control, other pregnant rats were fed the same liquid diet except that the ethanol was replaced by isocaloric sucrose (Pf). Each Et and Pf group was subdivided into two groups; one of the groups was treated with 1 mg/kg (i.p.) of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), an agonist for 5-HT2A/2C receptors, during GDs 13 to 19 (Et-DOI or Pf-DOI), and another was injected with saline vehicle only (Et-Sal or Pf-Sal). Their fetuses were removed by cesarean section on GD 19 or 20, and fetal brains were collected. An immunohistological examination of 5-HTergic neurons in the fetuses on embryonic day 20 using an antibody against tryptophan hydroxylase revealed that the number of 5-HTergic neurons in the midbrain raphe nuclei was significantly reduced in the Et-Sal fetuses compared to that of the Pf-Sal and Pf-DOI fetuses, whereas there were no significant differences between Et-DOI and each Pf control. Thus, we concluded that the reduction of 5-HTergic neurons that resulted in prenatal ethanol exposure could be alleviated by the enhancement of signaling via 5-HT2A/2C receptors during the fetal period

Ca2＋チャンネルビョウ マウス ニオケル ショウノウ ノ イジョウ ト ウンドウ シッチョウ

This review summarizes recent studies on the morphological abnormalities of cerebella in four ataxic mutant mice, i.e., tottering mouse, leaner mouse, rolling mouse Nagoya (RMN) and rocker mouse. These mutants carry mutations in the Ca2+ channel α1A subunit gene, and become useful models for human Ca2+ channelopathy such as episodic ataxia type-2 and familial hemiplegic migraine. Abnormal expression of tyrosine hydroxylase (TH) in some Purkinje cells has been observed in tottering mice, leaner mice and RMN, but not in rocker mice. However, Purkinje cells did not seem to synthesize catecholamines. Since the transcription of the TH gene is facilitated by Ca2+, TH expression in the mutant Purkinje cells indicates functional abnormality by alterations in intracellular Ca2+ concentrations. Corticotropin-releasing factor (CRF) immunoreactivity in some climbing or mossy fibers was higher in RMN than in controls. Double immunostaining for CRF and TH revealed a correspondence in the distribution of TH-positive Purkinje cells to terminal fields of CRF-positive climbing fibers in RMN. Therefore, CRF seems to alter granule and Purkinje cell functions, such as abnormal TH expression, indicating the possible expression of ataxic symptoms

Spinal nerve defects by prenatal VPA

To examine in detail spinal nerve defects induced by prenatal exposure to valproic acid in mice, pregnant ICR mice were subcutaneously injected with a single dose of 400 mg/kg valproic acid on gestational day 6, 7, 8, or 9, and their embryos were observed on gestational day 10. The whole-mount immunostaining using an anti-neurofilament antibody allowed us to identify spinal nerve defects, such as a loss of bundle, anastomosis among bundles arising from adjacent segment, and a disrupted segmental pattern of the dorsal root ganglia, in valproic acid-exposed embryos. The prevalence of spinal nerve defects was the highest in the embryos exposed to valproic acid on gestational day8 among the experimental groups. Then, effects of the administration dose of valproic acid on the prevalence of spinal nerve defects were examined on gestational day 10 and found to be dose-dependently increased. It was noteworthy that all embryos exposed to 600 mg/kg of valproic acid on gestational day 8 suffered spinal nerve defects. Folic acid (3 mg/kg/day) supplementation during gestational day 6 to 10 suppressed the prevalence of valproic acid-induced neural tube defects, which are common malformations in offspring prenatally exposed to valproic acid, but not that of spinal nerve defects.Thus, the spinal nerve defects due to prenatal valproic acid exposure might be induced by mechanisms different from those of neural tube defects. Because spinal nerve defects were predicted to be caused by the disrupted segmental arrangement of the somites and/or that of neural crest cells which was the origin of the dorsal root ganglia and/or abnormal polarity of the somite, this mouse model with spinal nerve defects at high incidence, would be useful to examine the effects of valproic acid on somitogenesis and morphogenesis of somite-associated structures