Cloning, expression and purification of cold adapted acetate kinase from Shewanella species AS-11

A psychrotrophic bacterium, Shewanella sp. AS-11 was isolated from a buccinid (shell) Neobuccinum living in the Antarctic ice-covered sea. An open reading frame of 1203 bp, coding for acetate kinase gene, called ack, was amplified, cloned into the expression vector, pETY-16b, and the enzyme was overproduced by using T7 system in Escherichia coli BL21 (DE3). After extraction of crude recombinant acetate kinase, the desired enzyme was able to be purified on a Blue Sepharose CL-6B and Super-Q affinity column chromatography. The molecular mass of the enzyme is about 86 kDa, which is associated with two monomers. In respect of pH, the enzyme was stable between 6 to 8 and maximum activity was obtained at 7.5. The purified enzyme was stable at 30°C but ligand bound enzyme was stable at 40°C. The structural comparison to mesophilic and thermophilic acetate kinases demonstrates that the psychrophilic one contains lower number of salt bridges and cation-pi interaction. So, it can be suggested that the enzyme is cold adapted with thermolabile and flexible structure.Keywords: Acetate kinase, thermolabile, cold adapted, flexible, activityAfrican Journal of Biotechnology Vol. 11(29), pp. 7454-7463, 10 April, 201

The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H\u3csub\u3e2\u3c/sub\u3e0\u3csub\u3e2\u3c/sub\u3e and plays an integral role in insulin signal transduction

Insulin stimulation of target cells elicits a burst of H2O2 that enhances tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins as well as distal signaling events in the insulin action cascade. The molecular mechanism coupling the insulin receptor with the cellular oxidant-generating apparatus has not been elucidated. Using reverse transcription-PCR and Northern blot analyses, we found that Nox4, a homolog of gp91phox, the phagocytic NAD(P)H oxidase catalytic subunit, is prominently expressed in insulin-sensitive adipose cells. Adenovirus-mediated expression of Nox4 deletion constructs lacking NAD(P)H or FAD/NAD(P)H cofactor binding domains acted in a dominant-negative fashion in differentiated 3T3-L1 adipocytes and attenuated insulin-stimulated H2O2 generation, insulin receptor (IR) and IRS-1 tyrosine phosphorylation, activation of downstream serine kinases, and glucose uptake. Transfection of specific small interfering RNA oligonucleotides reduced Nox4 protein abundance and also inhibited the insulin signaling cascade. Overexpression of Nox4 also significantly reversed the inhibition of insulin-stimulated IR tyrosine phosphorylation induced by coexpression of PTP1B by inhibiting PTP1B catalytic activity. These data suggest that Nox4 provides a novel link between the IR and the generation of cellular reactive oxygen species that enhance insulin signal transduction, at least in part via the oxidative inhibition of cellular protein-tyrosine phosphatases (PTPases), including PTP1B, a PTPase that has been previously implicated in the regulation of insulin action

A study of a karate trial teaching class in a teacher training course − based on students’ formative assessment

[EN] The purpose of this study was to examine the effectiveness of a karate trial teaching class in an initial teacher training course, through students’ formative assessment. It involved two case studies of trial teaching classes of karate and that of two other activities, taught by the students of an initial teacher training course. The results were assessed using the Students’ Formative Assessment of Physical Education (P.E.) Classes scale. Results showed significant differences between groups in “New discovery” (p<.05) and a trend toward statistical significance in “Skill growth”, “Fun Exercise” and “Learning friendly” (p<.10) based on the classes provided by karate and other teaching materials. This implies that karate might have different acute effects on students’ learning process in the context of school-level physical education

Secondary EML4?ALK-positive Lung Adenocarcinoma in a Patient Previously Treated for Acute Lymphoblastic Leukemia in Childhood: A Case Reportated for Acute Lymphoblastic Leukemia in Childhood: A Case Report

It is widely recognized that the risk of secondary neoplasms increases as childhood cancer survivors progress through adulthood. These are mainly hematological malignancies, and recurrent chromosome translocations are commonly detected in such cases. On the other hand, while secondary epithelial malignancies have sometimes been reported, chromosome translocations in these epithelial malignancies have not. A 33-year-old man who had been diagnosed with acute lymphoblastic leukemia and treated with chemotherapy almost 20 years earlier was diagnosed with lung adenocarcinoma. After chromosomal rearrangement of echinoderm microtubule- associated protein-like 4 gene and the anaplastic lymphoma kinase gene was detected in this adenocarcinoma, he responded to treatment with crizotinib. It was therefore concluded that this echinoderm microtubule-associated protein-like 4 gene-anaplastic lymphoma kinase gene-positive lung adenocarcinoma was a secondary epithelial malignancy

中学校武道領域における空手道授業に関する研究 ―教員養成課程の模擬授業の検討を通して―

The purpose of this study was to examine the effectiveness of a Karate trial teaching class in an initialteacher training course, through the student's formative assessment.　It involved two case studies　of trial teaching classes of Karate and that of two other activities, taught by the students of an initial teacher training course.　The results were assessed using the Student's Formative Assessment of Physical Education Classes scale developed by Takahashi et.al(1994).　Results of the study indicate that the students' formative assessment shows were significantly different (“new discovery”: p＜.05) and show a trend toward statistical significance (‘Skill growth': p＜.10, ‘Fun Exercise': p＜.10, ‘Learning friendly': p＜.10) based on the classes provided by Karate and other teaching materials.　This implies that Karate might have different effects on the learning of students compared to other activities as teaching materials in school-level physical education

Diffusion-weighted MR imaging in gynecologic cancers

Diffusion-weighted imaging (DWI) reflects changes in proton mobility caused by pathological alterations of tissue cellularity, cellular membrane integrity, extracellular space perfusion, and fluid viscosity. Functional imaging is becoming increasingly important in the evaluation of cancer patients because of the limitations of morphologic imaging. DWI is being applied to the detection and characterization of tumors and the evaluation of treatment response in patients with cancer. The advantages of DWI include its cost-effectiveness and brevity of execution, its complete noninvasiveness, its lack of ionizing radiation, and the fact that it does not require injection of contrast material, thus enabling its use in patients with renal dysfunction. In this article, we describe the clinical application of DWI to gynecological disorders and its diagnostic efficacy therein

A phase II study of amrubicin and carboplatin for previously untreated patients with extensive-disease small cell lung cancer

Background: Amrubicin is active in the treatment of extensive-disease small cell lung cancer (ED-SCLC), and carboplatin is an analogue of cisplatin with less non-hematological toxicity. Purpose: The purpose of this study was to determine the efficacy and toxicity of amrubicin and carboplatin combination chemotherapy for previously untreated patients with ED-SCLC. Patients and methods: Eligibility criteria were chemotherapy-naive ED-SCLC patients, performance status 0-1, age ?75, and adequate hematological, hepatic and renal function. Based on the phase I study, the patients received amrubicin 35 mg/m2 i.v. infusion on days 1, 2, and 3, and carboplatin AUC 5 i.v. infusion on day 1. Four cycles of chemotherapy were repeated every 3 weeks. Results: Thirty-five patients were enrolled, and 34 patients were eligible and assessable for response, toxicity, and survival. Patients\u27 characteristics were as follows: male/female = 26/8; performance status 0/1 = 4/30; median age (range) = 64 (41-75); stage IV = 34. Evaluation of responses was 6 complete response, 21 partial response, and 7 stable disease (response rate 79.4 %, 95 % CI 63.6-88.5 %). Grade 3 and 4 leukopenia, neutropenia, and thrombocytopenia occurred in 59, 82, and 26 %, respectively. There were no treatment-related deaths or pneumonitis. Three patients experienced hypotension as an amrubicin infusion reaction. The median progression-free survival time was 6.5 months. The median overall survival time and 1-, 2-, and 3-year survival rates were 15.6 months, and 63, 28, and 7 %, respectively. Conclusions: Amrubicin and carboplatin were effective and tolerable as chemotherapy for previously untreated patients with ED-SCLC. Further investigation of amrubicin and carboplatin is warranted

アミノ酸変異及びX線結晶解析を用いたリゾチームの安定性と変性の遷移状態の解析

GERERAL INTRODUCTION CHAPTER I Correlation Between the Differences in the Free Energy Change and Conformational Energy in the Folded State of Hen Lysozymes with Gly-Pro and Pro-Gly Sequences Introduced to the Same Site CHAPTER II Analysis of the Transition State in the Unfolding of Hen Lysozyme by Introduction of Gly-Pro and Pro-Gly Sequences at the Same Site CHAPTER III Analysis of the Stabilization of Hen Lysozyme with the Helix Microdipole and charged Side Chains CHAPTER IV Influence of Mutations of not Conserved N-Cap Residue,Gly4,on Stability and Structure of Hen Lysozyme CONCLUSIONMade available in DSpace on 2012-09-06T02:51:16Z (GMT). No. of bitstreams: 1 motoshima.pdf: 14085243 bytes, checksum: e6ca814a9b4ebee5d7a95ba8943830ff (MD5) Previous issue date: 1996-03-2