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molecular cloning and characterization of human age-related NADH oxidase (arNOX) proteins as members of the Tm9 superfamily of transmembrane proteins

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Advances in Biological Chemistry 2013年第2期3卷 187-197页

作者： Xiaoyu Tang Debby Parisi Bradley Spicer dorothy m. morré D. James morré mor-NuCo Inc. Purdue Research Park West Lafayette USA

Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the Tm9 family of transmembrane proteins. Peptide antisera raised to all five members of the human Tm9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog Tm9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known Tm9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the Tm9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.

关键词： Age-Related NADH Oxidase (arNOX) Tm-9 Superfamily of Transmembrane Proteins molecular Cloning Human Serum Plasma and Body Fluids Saliva

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World Journal of Cardiovascular Diseases 2014年第3期4卷 119-129页

作者： D. James morré Dale Kern Christiaan meadows Helen Knaggs dorothy m. morré morNuCo Inc. Purdue Research Park West Lafayette USA NuSkin International Provo USA

Age-related Ecto-Nicotinamide Adenine Dinucleotide Oxidase Disulfide Thiol Exchangers 3 (ENOX3) or age-related NADH oxidases (arNOX) are expressed at the cell surface as five members of the Tm-9 superfamily, initially membrane anchored, all functionally similar, with the N-termini exposed at the cell’s exterior. ECTO-NOXes are cell surface proteins with both time-keeping CoQH2 [NAD(P)H] oxidase and protein disulfidethiol interchange activities. They are designated as ECTO-NOX proteins because of their localization on the outer surface of the plasma membrane and to distinguish them from the phox-NOXes of host defense. A ca. 30 kDa N-terminal fragment is cleaved and accumulates in body fluids (serum, saliva, urine, perspiration). arNOXes appear around age 30 and increase steadily thereafter. Reduced quinones, i.e., reduced coenzyme Q, of the plasma membrane are natural substrates. NAD(P)H is oxidized as an artificial substrate. In one phase of the arNOX cycle electrons are transferred to oxygen to generate superoxide. Substrates for the shed forms of arNOX appear to be proteins of body fluids. Circulating lipoproteins and skin matrix proteins emerge as potentially important health-related targets. Through oxidation of collagen, elastin and other proteins of the skin matrix, arNOXes are major contributors to skin aging through tyrosine and thiol oxidation and subsequent cross linking. The main destructive action of arNOX, however, may be to directly oxidize circulating lipoproteins. arNOX in the blood is structured as an integral component of the LDL particle through site-specific binding. As such, arNOXes are implicated as major risk factors for cardiovascular disease due to specific oxidation of LDLs. The superoxide produced and its conversion to hydrogen peroxide would be one part of the potentially destructive properties by contribution to lipid oxidation. Inhibition of arNOX proteins provides a rational basis for anti-aging interventions and their elimination as a

关键词： Atherosclerosis Cardiovascular Risk Age-Related NADH Oxidase (arNOX) Tm9 Superfamily of Transmembrane Proteins Serum and Body Fluids Saliva Skin Aging Low Density Lipoproteins

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Ultradian oscillators of the circadian clock in Saccharomycescerevisiae

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Advances in Biological Chemistry 2013年第1期3卷 59-69页

作者： Sara S. Dick Aya Ryuzoji dorothy m. morré D. James morré Department of Foods and Nutrition Purdue University West Lafayette USA Department of medicinal Chemistry Purdue University West Lafayette USA

The yeast, Saccharomyces cerevisiae, has an ENOX1 activity with a period length of 24 min similar to that of other eukaryotes. In contrast to other eukaryotes, however, Saccharomyces cerevisiae has a second ENOX1-like activity with a period length of 25 min. The latter is distinguishable from the traditional ENOX1 on the basis of the longer period length along with resistance to an ENOX1 inhibitor, simalikalactone D, and failure to be phased by melatonin. In addition, two periods are apparent in measurements of oxygen consumption indicating that the consumption of oxygen to water occurs independently by homodimers of both of the two forms of ENOX. Based on the measurements of glyceraldehyde-3- phosphate dehydrogenase, S. cerevisiae exhibits circadian activity maxima at 24 and 25 h together with a 40 h period possibly representing the 40 min metabolic rhythm of yeast not observed in our measurement of oxygen consumption and normally observed only with continuous cultures. The findings are indicative of at least three independent time-keeping systems being operative in a single cell.

关键词： Biological Clock ECTO-NOX (ENOX) Proteins Ultradian Oscillator Yeast (Saccharomyces cerevisiae)

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Identification of the constitutive ultradian oscillator of the circadian clock (ENOX1) in Saccharomyces cerevisiae

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Advances in Biological Chemistry 2013年第3期3卷 320-328页

作者： Sara S. Dick Aya Ryuzoji dorothy m. morré D. James morré Department of Foods and Nutrition Purdue University West Lafayette USA mor-Nu Co. LLC Purdue Research Park West Lafayette USA

A yeast (Saccharomyces cerevisiae) deletion library was screened based on NADH fluorescence using a 384 well plate assay and robotics to identify a yeast isolate lacking the 24 min periodic cell surface oxidase. The oxidase was shown previously to be a candidate ultradian oscillator of the yeast’s biological clock. The cDNA was cloned from a yeast overexpression library and the encoded protein was expressed in bacteria and characterized. Glyceraldehyde-3-phosphate dehydrogenase activity was used as the cellular circadian indicator. The identified gene was YmL117W which encodes a ca 126 kDa putative RNA-binding protein. The candidate ENOX1 activity from yeast had functional characteristics similar to those of other constitutive ENOX1 proteins of eukaryotes exhibiting oscillating activities with a temperature independent period length of 24 min phased by melatonin and low frequency electromagnetic fields and susceptible to inhibition by the ENOX1 inhibitor, simalikalactone D. The YmL117W deletion mutant cells lacked the ENOX1 clock output present in wild type yeast. The findings identify YmL117W as the ENOX1 of Saccharomyces cerevisiae and support its proposed function as an ultradian oscillator of the yeast biological clock.

关键词： Biological Clock ECTO-NOX (ENOX) Proteins Ultradian Oscillator Yeast (Saccharomyces cerevisiae)

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Application of stereological methods to investigate the healthy or injured brain:Studies on the healthy basal ganglia and on three different animal models that are relevant to either cerebral palsy,ADHD-like hyperactivity or schizophrenia

Application of stereological methods to investigate the heal...

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第十六届中国体视学与图像分析学术会议——交叉、融合、创新

作者： dorothy E.OORSCHOT Department of Anatomy and the Brain Health Research CentreUniversity of Otago

Purpose:The application of stereological methods is critical to advancing knowledge on the healthy or the injured *** this plenary presentation,examples from my laboratory of the scientific value of stereological methods will be ***,examples will be presented of:*** the definitive measure of the absolute number of neurons is preferable to using indirect measures such as neuronal density or the volume of a brain *** topic is relevant to the following scientific questions:Is there neuronal loss after injury to the brain?Is a specific treatment effective at rescuing neurons in an animal model of cerebral palsy?Is anatomical convergence at the gross anatomical level reflected in the absolute number of neurons within the normal,healthy basal ganglia?*** measurement of the amount of myelin in the brain and how this can be correlated with behavioural measures,such as the hyperactivity that is relevant to attention deficit hyperactivity disorder(ADHD),in the same *** measurement,at the nanosale level,of identified inhibitory synapses in the brain and how this reveals differences in an animal model of ADHD-like hyperactivity versus an animal model of *** and methods:For the first topic,serial 40 μm Technovit sections were cut through the entire striatum,or basal ganglia,of Sprague-Dawley rats.A systematic series of sections,after a random start,were stained with cresyl *** Cavalieri and optical disector stereological methods were used to measure the absolute number of neurons,N,within these brain ***,the Cavalieri method was used to measure the total(absolute) reference volume(Vref) of the brain structure of *** optical disector method was used to measure the number of neurons in a known subvolume(Nv,*** neuronal density).To obtain N,the Vref was multiplied by the *** the second topic,Cavalieri's method was combined with stereological measurement of

关键词： Cavalieri's method optical disector method absolute quantities brain injury neuroscience

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环境因素对螺旋藻的生长和生化成分影响的分析

环境因素对螺旋藻的生长和生化成分影响的分析

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作者： NYABUTO dorothy KEmUmA 南京农业大学

学位级别：硕士

螺旋藻被广泛应用于人们生活的方方面面,如卫生、食品和化妆品行业等。本论文通过改变不同的物理和化学因素水平,即不同pH,纳米氧化镁(mgO),氮源,磷源,碳源,盐浓度和生长培养基等,对螺旋藻的生物量,叶绿素和蛋白质的生产条件进行优化,... 详细信息

螺旋藻被广泛应用于人们生活的方方面面,如卫生、食品和化妆品行业等。本论文通过改变不同的物理和化学因素水平,即不同pH,纳米氧化镁(mgO),氮源,磷源,碳源,盐浓度和生长培养基等,对螺旋藻的生物量,叶绿素和蛋白质的生产条件进行优化,达到优化生物量的目的。实验过程中螺旋藻的生长温度30±2℃,光强为44001ux,光/暗周期为14:10 h。本论文探讨了不同环境因素水平对螺旋藻培养生理生化指标的影响,主要结果如下:培养基中pH=9时,螺旋藻的干重、蛋白质和叶绿素a含量最高,分别为7.83±0.29g/L,1.34±0.12mg/mL,18.64±0.06μg/mL。当 mgO 的浓度为 0.8mm 时,最大干重、蛋白质和叶绿素含量分别为 3.48±3.48g/L,3.44±0.312mg/mL,4.80±0.12μng/mL。以 NaNO3 作为氮源时,获得螺旋藻的干重为2.24±0.13g/L,而蛋白质和叶绿素a含量分别为3.24±0.30mg/mL和2.53±0.24μg/ml。以K2HPO4作为磷源时,获得最大的干重为2.08±0.17g/10ml,蛋白质和叶绿素含量分别为 2.93±0.06mg/ml,2.52±0.21μg/ml。NaHCO3作为碳源时,最高干重为1.18±0.02g/L,蛋白质含量为2.97±0.10mg/ml,叶绿素a含量为1.15±0.11μg/ml。采用通用Zarrouk培养基时培养时,干重为3.48±0.34 g/L,蛋白质含量为5.26±0.41mg/ml,叶绿素a含量为5.18±0.43μg/ml。改变培养基的盐浓度,发现当盐浓度为2.5%时,获得最大干重为1.44±0.06g/L,蛋白质含量为117.77±2.00μg/ml,叶绿素a含量为1.86±0.13mg/L,且获得了最高细胞生长速率0.22±0.01μm/天。本研究结果可为大规模生产高蛋白质含量、极具发展前途的健康食品螺旋藻提供理论依据。

关键词：生物量叶绿素a 蛋白质螺旋藻

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葛根素对急性心肌缺血狗区域性心肌血流与心脏血流动力学的作用

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药学学报 1984年第11期19卷 801-807页

作者：范礼理,D D O'keefe,W J Powell 中国医学科学院药物研究所 The Departments of medicine and Surgery of the massachusetts General Hospital and Harvard medical School Boston m A.02114 USA The Departments of medicine and Surgery of the massachusetts General Hospital and Harvard medical School Boston m A.02114 USA Jr 北京

麻醉开胸狗ⅳ葛根素(puerarin)可减慢心率(HR)、降低主动脉压(mAP),用同位素标记微球法测得的缺血区侧枝冠脉血流量并不减少。从狗的右室旁路制备的心脏血流动力学实验发现葛根素明显减低张力一时间指数(TTI)与左室压力升高速度(LV dp/... 详细信息

麻醉开胸狗ⅳ葛根素(puerarin)可减慢心率(HR)、降低主动脉压(mAP),用同位素标记微球法测得的缺血区侧枝冠脉血流量并不减少。从狗的右室旁路制备的心脏血流动力学实验发现葛根素明显减低张力一时间指数(TTI)与左室压力升高速度(LV dp/dt)。当mAP调整到给药前的水平时,TTI与LV dp/dt恢复,进入缺血区的侧技血流增加,非缺血区的冠脉血流量(CBF)亦增加。葛根素减低冠脉血管阻力(CVR)的作用比减低全身血管阻力(SVR)的作用更显著。葛根素不影响心肌收缩力,但增加局部心肌缺血时的侧枝血流并减少与心肌氧消耗有关的血流动力学参数。这些结果提示葛根素有益于治疗心肌缺血。

关键词：葛根素心肌缺血放射性微球区域性心肌血流心脏血流动力学

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安全查询处理策略

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计算机工程与科学 1991年第1期13卷 18-30页

作者： T.F.keefe m.B.Thuraisingham W.T.Tsai 范宏深

本文介绍了几种技巧,可以使权限级(clearance level)不相同的用户在不违反数据库管理系统安全性的情况下,访问各个敏感级的数据。

关键词：数据库安全查询数据处理

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molecular Cloning and Characterization of a Candidate ENOX Protein of Saccharomyces cerevisiaewith a 25 min Period Insensitive to Simalikalactone D Inhibition and melatonin

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Advances in Biological Chemistry 2014年第5期4卷 339-350页

作者： Aya F. Ryuzoji Debby H. Parisi Sara Dick James Kim dorothy m. morré D. James morré Department of Biology Valparaiso University Valparaiso USA mor-NuCo Inc. Purdue Research Park West Lafayette USA

A yeast deletion library was screened based on NADH fluorescence using a 384-well plate assay to identify a yeast isolate lacking a previously identified cell surface oxidase exhibiting an oscillatory pattern with a period length of 25 min and resistant to the ENOX1-specific inhibitor simalikalactone D (YNOX for yeast-specific ENOX = ENOX4). The cDNA was cloned from a yeast over expression library using NADH fluorescence analyzed by Fast Fourier transform and decomposition fits. The objective was to identify and sequence an ENOX homologue in Saccharomyces cerevisiae with a 25 min rather than a 24 min period length (YNOX). The finding identified YDR005C as the yeast ENOX protein with a temperature-independent 25 min period length and insensitive to inhibition by simalikalactone D. The encoded protein was expressed in bacteria and characterized. Gel slices corresponding to 55 kDa and 39 kDa His-tagged proteins exhibited 25 min oscillatory patterns not inhibited by 1 μm simalikalactone D for both NADH oxidation and reduced coenzyme Q10 oxidation as well as a protein disulfide-thiol interchange activity which alternated with the oxidative activities. Activities were phased by low-frequency electromagnetic fields but, in contrast, to yeast ENOX1, not by addition of melatonin. The assay in the presence of D2O shifted the length of the oscillatory period from 25 min to 32 min. The YDR005C deletion mutant cells lacked the ENOX4 clock output present in the wild type yeast.

关键词： Biological Clock ECTO-NOX (ENOX) Proteins YNOX = ENOX4 Ultradian Oscillator Yeast (Saccharomyces cerevisiae)

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Reimagining the Visceral Protective Layer with Tailored manipulation: A Case Report

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Surgical Science 2022年第2期13卷 53-65页

作者： R. Dewayne Edwards Luis G. Fernandez Sean O’keefe michelle Baribault marc R. matthews Burn Surgery and Surgical Critical Care The Arizona Burn Center Phoenix AZ USA Department of Surgery University of Texas Health Science Center Tyler TX USA 3m Phoenix AZ USA mountain Vista medical Center Mesa AZ USA The Arizona Burn Center Phoenix AZ USA

The visceral protective layer is a standard component of the ABTHERATm systems for temporary abdominal closures. Nonetheless, there are circumstances where the standard, fenestrated visceral protective layer is too large to be successfully applied into every patient’s open abdomen, such as within the abdomen of a child, smaller adult or a patient with previously placed ostomies or drains. The fenestrated, visceral protective layer may require alterations or tailoring for adequate deployment instead of placing the bulk of the visceral protective layer entirely into the open abdomen for temporary abdominal closure. This case report illustrates how the visceral protective layer can be adapted or “reimagined” to conform to a patient with unique or complex abdominal domain features when utilizing the ABTHERATm device prior to facial closure or abdominal wall reconstruction. Photographs are utilized in a step-by-step fashion to aid the clinician in these detailed maneuvers.

关键词： Open Abdomen Temporary Abdominal Closure Visceral Protective Layer Loss of Abdominal Domain

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