遺伝子レベルからの中枢神経機能のアプローチ-オプシンm-RNAの解析-

金沢大学がん研究所研究課題/領域番号:56770116, 研究期間(年度):1981出典：「遺伝子レベルからの中枢神経機能のアプローチ-オプシンm-RNAの解析-」研究成果報告書　課題番号56770116（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-56770116/）を加工して作

網膜に特異的な遺伝子のクローニングとその応用

金沢大学がん研究所我々が網膜と脳との差(プラスーマイナス法)を利用して単離したMEKAcDNAクローンは、コンピューター解析から既知物質と類似性を示さない新しい網膜に特異的なMEKA蛋白質をコードしていることがわかった。MEKA蛋白質(分子量;27KDa,等電点;5.4)の生理機能を知るために以下の実験を行った。1.抗MEKAの作製遺伝子操作法を用いてMEKA融合蛋白質を大腸菌内で大量に合成させた。すなわち発現ベクターPEX1がコートするβーgalactosidaseC末端にMEKAcDNAの断片を組み込んだ。SDS-PAGEでMEKA融合蛋白質を分離、抽出してウサギに免疫した。2.MEKA抗体を用いた実験抗MEKAを用い種々の動物網膜組織との組織化学、及びウエスターンブロットの解析からMEKA蛋白質は、網膜組織に特異的に発現していること、またその局在が光受容細胞にのみ認められた。正常マウスと網膜変性rdマウスを用いた加令および網膜変性過程での組織化学も行った。現在ウシ網膜からMEKA蛋白質を約30%の純度で精製しており、単一バンドまでの精製を試みている。3.MEKAゲノムの単離組織に特異的な遺伝子の発現を解析するためには、そのゲノムの単離が必要であることから我々はヒトMEKAゲノムを単離した。現在ウシMEKAゲノムの単離とこれら遺伝子の発現機序の解明を試みている。We have isolated a MEKA cDNA clone by differential colony hybridization method. The MEKA cDNA encodes unknown soluble protein whose molecular weight and pI are calculated to be 27KDa and 5.35, respectively. We found by Northern and in situ hybridization that MEKA mRNA was transcribed only in the photoreceptor cells. The MEKA cDNA was ligated with expression vector PEX1 and a MEKA-fusion protein synthesized in E.coli was purified and used as antigen. By the Western blot and immunohistochemical study, we found that MEKA protein is expressed in the photoreceptor cells, but not in other tissues.研究課題/領域番号:62571022, 研究期間(年度):1987 – 1988出典：研究課題「網膜に特異的な遺伝子のクローニングとその応用」課題番号62571022（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-62571022/625710221988kenkyu_seika_hokoku_gaiyo/）を加工して作

Surgical treatment for thyrotoxic hypokalemic periodic paralysis: case report

Thyrotoxic hypokalemic periodic paralysis (THPP) is a rare, potentially life-threatening endocrine emergency. It is characterized by recurrent muscle weakness and hypokalemia. Because many THPP patients do not have obvious symptoms and signs of hyperthyroidism, misdiagnosis may occur. The published studies revealed that definitive therapy for THPP is control of hyperthyroidism by medical therapy, radioactive iodine or surgery, but the long-term post-operative follow-up result was not observed. We reported two cases of medically refractory THPP with recurrent paralysis of extremities and hypokalemia, and both were combined with thyroid nodules. Both patients were treated with total thyroidectomy; the pathology revealed that one is Graves' disease with thyroid papillary carcinoma, and the other is adenomatous goiter with papillary hyperplasia. No episode of periodic paralysis was noted and laboratory evaluation revealed normal potassium level during the post-operative follow up. Our experience suggests that total thyroidectomy by experienced surgeon is an appropriate and definite treatment for medically refractory THPP, especially in cases combined with thyroid nodules

Secret lifestyles of Neurospora crassa

Peer reviewe

Factors Influencing the Competency of Head Nurses When Assisting With Inhospital Cardiopulmonary Resuscitation

AbstractObjectiveHead nurses in a hospital setting work to maintain the quality of nursing care and ensure that staff nursing skills are up-to-date. Although cardiopulmonary resuscitation (CFR) is most often performed by physicians in a hospital, nurses play crucial assistance roles, including the administration of medication, record-keeping, obtaining intravenous access, and helping with intubation. Patient outcome is therefore related to nurses' skill level in CFR. We hypothesized that the more senior head nurses would be more skillful at assisting with CFR.Materials and MethodsWe conducted an observational study of 55 head nurses, who were asked to perform a 4-minute simulation in which they assisted with CFR using a resuscitation manikin. We collected demographic data, including age and years since becoming a nurse and becoming a head nurse, together with a stress score, which assessed the stress felt by the nurses while taking the test. These measures were analyzed along with the accuracy of the nurses' skills while performing CFR.ResultsTwenty-two skills were evaluated, with each skill graded as either a pass or fail. Only 30/55 (54.5%) of the head nurses passed all 22 skills. There was a significant difference in the numbers of skills with a pass rating (p = 0.043) in relation to the units where the head nurses were employed. Their performance also showed a strong negative correlation with age (p = 0.001), work experience (p = 0.028), and time since becoming head nurse (p<0.001). However, there was no significant difference between the numbers of skills with a pass rating and pre-test stress score (p = 0.129).ConclusionIn our study, the head nurses of surgical wards, medical wards, and intensive care units had better scores than the head nurses of pediatric, obstetrics-gynecology, and other units. In contrast with our original hypothesis, we found that the more senior head nurses' skills when assisting with CFR were poorer than those of their more junior colleagues. This suggests that senior head nurses should undergo frequent refresher courses, not only to help maintain their life-saving abilities, but also so that they can better mentor less experienced nurses in situations requiring CFR

YC-1 [3-(5Ј-Hydroxymethyl-2Ј-furyl)-1-benzyl Indazole] Inhibits Neointima Formation in Balloon-Injured Rat Carotid through Suppression of Expressions and Activities of Matrix Metalloproteinases 2 and 9

ABSTRACT Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, and postrevascularization production of vascular smooth muscle cells may play key roles in development of arterial restenosis. We investigated the inhibitory effect of 3-(5Ј-hydroxymethyl-2Ј-furyl)-1-benzyl indazole (YC-1), a benzyl indazole compound, on MMP-2 and MMP-9 activity in a ballooninjury rat carotid artery model. Injury was induced by inserting a balloon catheter through the common carotid artery; after 14 days, histopathological analysis using immunostaining and Western blotting revealed significant restenosis with neointimal formation that was associated with enhanced protein expression of MMP-2 and MMP-9. However, these effects were dosedependently reduced by orally administered YC-1 (1-10 mg/ kg). In addition, gelatin zymography demonstrated that increased MMP-2 and MMP-9 activity was diminished by YC-1 treatment. On the other hand, YC-1 inhibited hydrolysis of the fluorogenic quenching substrate Mca-Pro-Leu-Gly-Leu-DpaAla-Arg-NH 2 by recombinant MMP-2 and MMP-9 with IC 50 values ϭ 2.07 and 8.20 M, respectively. Reverse transcription-polymerase chain reaction analysis of MMP-2 and MMP-9 mRNA revealed that YC-1 significantly inhibited mRNA levels of MMPs. Finally, for the YC-1 treatment group, we did not observe elevation of cGMP levels using enzyme-linked immunosorbent assay, suggesting that YC-1 inhibition of neointimal formation is not through a cGMP-elevating pathway. These data show YC-1 suppression of neointimal formation is dependent on its influence on MMP-2 and MMP-9 protein, mRNA expression, and activity, but not through a cGMP-elevating effect. YC-1 shows therapeutic potential for treatment of restenosis after angioplasty. During the past 20 years, one focus of cardiovascular pharmaceutical research has been the development of drugs that inhibit intimal hyperplasia. Despite many attempts, no clinical trial has proven that there is an effective pharmacological solution to the problem Matrix metalloproteinases (MMPs) are a family of structurally related zinc-endopeptidases that degrade components of extracellular matrix associated with vascular remodeling during vascular injury-induced neointima formatio

An ongoing process: A qualitative study of how the alcohol-dependent free themselves of addiction through progressive abstinence

<p>Abstract</p> <p>Background</p> <p>Most people being treated for alcoholism are unable to successfully quit drinking within their treatment programs. In few cases do we know the full picture of how abstinence is achieved in Taiwan. We tracked processes of abstinence in alcohol-dependency disorders, based on study evidence and results. This research explores the process of recovery from the viewpoint of the alcohol-dependent.</p> <p>Methods</p> <p>Semi-structured interviews were conducted in two different settings, using purpose sampling, during 2003-2004. The data were analyzed using content analysis. Participants were 32 adults, purposefully selected from an Alcoholics Anonymous group and a psychiatric hospital in North Taiwan.</p> <p>Results</p> <p>We found that the abstinence process is an ongoing process, in which the alcohol-dependent free themselves of addiction progressively. This process never ends or resolves in complete recovery. We have identified three stages in the struggle against alcoholism: the Indulgence, Ambivalence and Attempt (IAA) cycle, in which the sufferer is trapped in a cycle of attempting to give up and failing; the Turning Point, in which a Personal Nadir is reached, and the Ongoing Process of abstinence, in which a constant effort is made to remain sober through willpower and with the help of support groups. We also discuss Influencing Factors that can derail abstinence attempts, pushing the sufferer back into the IAA cycle.</p> <p>Conclusion</p> <p>This study provides important points of reference for alcohol and drug service workers and community healthcare professionals in Taiwan, casting light on the abstinence process and providing a basis for intervention or rehabilitation services.</p

SEPTIN12 Genetic Variants Confer Susceptibility to Teratozoospermia

It is estimated that 10–15% of couples are infertile and male factors account for about half of these cases. With the advent of intracytoplasmic sperm injection (ICSI), many infertile men have been able to father offspring. However, teratozoospermia still remains a big challenge to tackle. Septins belong to a family of cytoskeletal proteins with GTPase activity and are involved in various biological processes e.g. morphogenesis, compartmentalization, apoptosis and cytokinesis. SEPTIN12, identified by c-DNA microarray analysis of infertile men, is exclusively expressed in the post meiotic male germ cells. Septin12+/+/Septin12+/− chimeric mice have multiple reproductive defects including the presence of immature sperm in the semen, and sperm with bent neck (defect of the annulus) and nuclear DNA damage. These facts make SEPTIN12 a potential sterile gene in humans. In this study, we sequenced the entire coding region of SEPTIN12 in infertile men (n = 160) and fertile controls (n = 200) and identified ten variants. Among them is the c.474 G>A variant within exon 5 that encodes part of the GTP binding domain. The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12. Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm <14%) and their sperm showed bent tail and de-condensed nucleus with significant DNA damage. Ex vivo experiment showed truncated SEPT12 inhibits filament formation in a dose-dependent manner. This study provides the first causal link between SEPTIN12 genetic variant and male infertility with distinctive sperm pathology. Our finding also suggests vital roles of SEPT12 in sperm nuclear integrity and tail development

RRM adjacent TARDBP mutations disrupt RNA binding and enhance TDP-43 proteinopathy

Amyotrophic lateral sclerosis (ALS) presents with focal muscle weakness due to motor neuron degeneration that becomes generalized,leading to death from respiratory failure within 3–5 years from symptom onset. Despite the heterogeneity of aetiology, TDP- 43 proteinopathy is a common pathological feature that is observed in 495% of ALS and tau-negative frontotemporal dementia(FTD) cases. TDP-43 is a DNA/RNA-binding protein that in ALS and FTD translocates from being predominantly nuclear to formdetergent-resistant, hyperphosphorylated aggregates in the cytoplasm of affected neurons and glia. Mutations in TARDBP accountfor 1–4% of all ALS cases and almost all arise in the low complexity C-terminal domain that does not affect RNA binding andprocessing. Here we report an ALS/FTD kindred with a novel K181E TDP-43 mutation that is located in close proximity to the RRM1 domain. To offer predictive gene testing to at-risk family members, we undertook a series of functional studies to characterizethe properties of the mutation. Spectroscopy studies of the K181E protein revealed no evidence of significant misfolding.Although it is unable to bind to or splice RNA, it forms abundant aggregates in transfected cells. We extended our study to includeother ALS-linked mutations adjacent to the RRM domains that also disrupt RNA binding and greatly enhance TDP-43 aggregation,forming detergent-resistant and hyperphosphorylated inclusions. Lastly, we demonstrate that K181E binds to, and sequesters, wild-type TDP-43 within nuclear and cytoplasmic inclusions. Thus, we demonstrate that TDP-43 mutations that disrupt RNAbinding greatly enhance aggregation and are likely to be pathogenic as they promote wild-type TDP-43 to mislocalize andaggregate acting in a dominant-negative manner. This study highlights the importance of RNA binding to maintain TDP-43solubility and the role of TDP-43 aggregation in disease pathogenesis