Cloning and expression of a serine racemase gene homologue of the green alga Chlamydomonas reinhardtii and characterization of the gene product

A unicellular green alga Chlamydomonas reinhardtii (C. reinhardtii) has served as a model system to study many fundamental biological processes. We demonstrated that some D-amino acids have no inhibitory effect on the growth of C. reinhardtii and the green alga has alanine racemase and D-threonine aldolase. The homologous gene of serine racemase was found on the genome sequence of C. reinhardtii. In this study, a homologous gene of serine racemase on the genome of C. reinhardtii was cloned and expressed in E. coli cells, and the gene product was purified and characterized. Total RNA was extracted from C. reinhardtii cells. Sense and antisense primers were designed for PCR based on the upstream and downstream regions of the putative gene for serine racemase. First strand cDNA was synthesized from the mRNA and the antisense primer. Amplification of nucleotides between the two primers was performed with the cDNA. The fragment (ser-h) was sequenced. The deduced protein consisted of 340 amino acids with a molecular weight of 35,300. The amino acid sequence of the protein showed similarities to the reported serine racemases; Oryza sativa, 55%; Mus musculus, 52%; Schizosaccharomyces pombe, 39%. A modified serine racemase homologous (ser-h\u27) whose codons were optimized for E. coli was synthesized and used to construct pET24/ser-h\u27 and to transform BL21 (DE3). SDS-PAGE of the crude extract revealed that the gene product was overexpressed. The gene product was purified to electrophoretic homogeneity from the recombinant cells using ammonium sulfate fractionation and Column chromatography. Further characterization and crystallization of the enzyme are currently under study

Choroidal Folds in Acute-Stage Vogt-Koyanagi-Harada Disease Patients with Relatively Short Axial Length

Purpose: To report 2 cases of Vogt-Koyanagi-Harada disease accompanied by remarkable choroidal folds in the acute stage. The early indicator of recurrence in these 2 cases was the identification of choroidal folds by spectral-domain optical coherence tomography (SD-OCT). Case Reports: A 68-year-old woman (Case 1) presented with visual loss in both eyes. Funduscopic examination revealed optic disc swelling and serous retinal detachment in both eyes. SD-OCT revealed remarkable choroidal folds and serous retinal detachment. After the initiation of systemic steroid treatment, choroidal folds disappeared rapidly and the amount of serous retinal detachment reduced remarkably. Choroidal folds observed on SD-OCT were the early indicators of recurrence prior to the emergence of serous retinal detachment. A 62-year-old woman (Case 2) presented with bilateral blurred vision and metamorphopsia. SD-OCT showed remarkable choroidal folds and serous retinal detachment in both eyes. After the initiation of systemic steroid treatment, choroidal folds and serous retinal detachment disappeared. At the time of recurrence, choroidal folds were observed by OCT. Discussion: During monitoring of Vogt-Koyanagi-Harada disease treatment, choroidal folds could be an early sign of recurrence. When choroidal folds are observed and recognized as an early indicator of recurrence, a prompt increase in steroids can improve the patients’ prognosis. Finally, both cases presented here had relatively short axial lengths, and we speculate that a shortened axial length may be a cause of choroidal folds in the acute stage of the disease

Purification and pressure dependence of alanine racemase from the psychro- piezophilic bacterium shewanella violacea DSS 12

Shewanella violacea DSS12 (S. violacea) is a psychrophilic and piezophilic bacterium, isolated from mud of the Ryukyu Trench in Japan. The bacterium displays optimal growth at 8°C and 30 MPa. Alanine racemase is an enzyme which catalyses the interconversion of l-alanine and d-alanine, and is responsible for the synthesis of d-alanine contained in the peptidoglycan of bacterial cell wall. In this study, we purified alanine racemase from S. violacea and investigated the enzymological characteristics of alanine racemase. The bacterium was aerobically cultured using marine broth 2216 in a 5-liter medium bottle at 4°C for 3 days. The bacterial cells were lysed by applying of 100 MPa pressure using a French press, and the lysate was centrifuged. The supernatant obtained was ultracentrifuged at 141,000 g, and the supernatant obtained was applied to ammonium sulfate fractionation. The active fraction was dissolved and passed through a butyl-Toyopearl, phenyl-Sepharose, and shodex KW-200 columns to obtain a partially purified enzyme. Consequently, the enzyme was purified 540-fold and showed a specific activity of 2.68 μmol/min/mg. Alanine racemase exhibited high activity against l-Ala and l-Ser as substrates. The optimal pH and temperature of alanine racemase were 9.0 and 25°C, respectively. Please click Additional Files below to see the full abstract

A Case of Acute Posterior Multifocal Placoid Pigment Epitheliopathy Demonstrating Vogt-Koyanagi-Harada Disease-Like Optical Coherence Tomography Findings in the Acute Stage

Purpose: We report a case of acute posterior multifocal placoid pigment epitheliopathy (APMPPE), which was difficult to differentiate from posterior pole-type Vogt-Koyanagi-Harada (VKH) disease because the lesions were mainly located in the macula bilaterally. Case Report: A 33-year-old man presented with rapid bilateral loss of vision. Fundoscopy revealed yellow-white subretinal lesions in the posterior pole of both eyes. Optical coherence tomography (OCT) revealed the presence of subretinal fluid with a subretinal septum. After initiation of systemic steroids, OCT revealed that the amount of subretinal fluid decreased immediately. However, vision loss was less responsive to the therapy, and OCT revealed partial reorganization of the inner segment/outer segment (IS/OS) line in the bilateral macular areas after therapy. Discussion: In our case, the location of the macular lesions made it difficult to differentiate APMPPE from VKH disease by fluorescein angiography. OCT images showed VKH disease-like findings of serous retinal detachment with a subretinal septum. The outer nuclear layer disappeared and the IS/OS line in the affected area was disorganized in the acute stage of the disease. In this case, the rapid loss of vision was specific to the onset pattern of APMPPE, and the slow response to therapy was very different from the response typically observed in VKH disease. Thus, careful consideration of the clinical course is important for diagnosing APMPPE

Cognitive Decline Predicts Long-Term Care Insurance Requirement Certification in Community-Dwelling Older Japanese Adults: A Prospective Cohort Study

Aim: The purpose of this prospective cohort study is to examine whether cognitive decline is an independent predictor of new long-term care insurance (LTCI) requirement certifications in Japan. Methods: A total of 5,765 community-dwelling older Japanese adults who, at baseline, were independent in terms of their activities of daily living participated in this study and were followed up for 18 months. The outcome measure was the number of new LTCI requirement certifications during the 18-month period of the study. We collected demographic information through questionnaires and assessed cognitive skills with the Cognitive Performance Scale (CPS). The participants were divided into 3 groups according to CPS scores (0, 1, and 2 or greater). Results: During the 18-month period, 399 subjects (6.9%) became newly certified for LTCI services. In a multivariate Cox proportional hazards model, older participants with a CPS score of 1 (adjusted HR: 1.39, 95% CI: 1.08-1.77) and 2 or greater (adjusted HR: 2.27, 95% CI: 1.74-2.96) were significantly more likely to receive an LTCI certification compared to those with a CPS score of 0. Conclusions: Cognitive decline is an independent predictor of new LTCI requirement certifications and the severity of cognitive decline in elderly adults is positively associated with receiving an LTCI requirement certification in Japan

Shoe-fit is correlated with exercise tolerance in community-dwelling elderly people

[Purpose]: Maintenance of physical activity significantly affects quality of life, and the frequency of physical activity depends upon exercise tolerance. However, there is minimal information on the external factors that contribute to exercise tolerance. The aim of this study was to examine the association between exercise tolerance and shoe-fit in community-dwelling elderly people. [Methods]: Subjects were 155 elderly, healthy, community-dwelling Japanese volunteers. Exercise tolerance (Shuttle Walk Test [SWT]), 10-m walking time (10mWT), and forced expiratory volume in 1 second (FEV1) were measured. Shoe-fit was assessed and participants were divided into three groups according to the heel-fit of their shoes (Too Loose, Loose, Fit). Group scores in the above variables were compared. Further, a multivariate logistic regression model using a stepwise method was performed to investigate which shoe-fit factors were independently associated with SWT. [Results]: No significant differences in age, gender, body mass index, 10mWT, FEV1, or presence or absence of pain sites were observed between the three groups. The Fit (p = 0.001) and Loose (p = 0.008) groups had significantly higher SWT score than the Too Loose group. Multivariate logistic regression analysis showed that poor heel-fit was significantly correlated with a low SWT score, even following adjustments for age, gender, 10mWT and FEV1 (odds ratio: 0.25, 95% confidence interval: 0.07–0.95, p = 0.04). [Conclusions]: This study demonstrates that heel-fit is associated with exercise tolerance in community-dwelling elderly people. It is important for elderly people to wear adequate fit shoes in order to enhance physical functions and prevent declining physical functions