工业微生物中NADH的代谢调控

NADH是微生物代谢网络中的一种关键辅因子。调节微生物胞内NADH的形式与浓度是定向改变和优化微生物细胞代谢功能, 实现代谢流最大化、快速化地导向目标代谢产物的重要手段之一。以下在详尽总结了NADH生理功能的基础上, 从生化工程(添加外源电子受体、不同氧化还原态底物及NAD合成前体物, 调节培养环境和氧化还原电势)和代谢工程(过量表达NADH代谢相关酶、缺失NADH竞争途径及引入NADH外源代谢途径)两方面分析、归纳了NADH代谢调控策略, 进而凝练出调控NADH/NAD+比率调节微生物细胞代谢功能研究方面亟待解决的3个科学问题及可能的解决途径。     

高山被孢霉ATCC 32222中细胞色素b5还原酶I的克隆、表达和功能鉴定

【目的】鉴定产油微生物高山被孢霉ATCC 32222中细胞色素b_5还原酶Ⅰ的功能。【方法】将高山被孢霉ATCC 32222中膜结合细胞色素b_5还原酶Ⅰ基因与人可溶性细胞色素b_5还原酶基因序列比对,去除该基因N端穿膜区域后,与人可溶性细胞色素b_5基因分别在大肠杆菌中异源表达;通过钴离子亲和层析、离子交换和分子排阻色谱等方法对表达产物进行纯化;以2,6-二氯靛酚钠(DCIP)为底物,测定细胞色素b_5还原酶Ⅰ的体外活性及其对NADH和NADPH的偏好性;在反应体系中存在NADH时,通过全波长扫描方法检测细胞色素b_5还原酶Ⅰ与细胞色素b_5的相互作用。【结果】高山被孢霉ATCC 32222中膜结合细胞色素b_5还原酶Ⅰ被成功可溶表达,经纯化后检测到体外活性:使用NADH时酶活为564.57 U,使用NADPH时为51.97 U;在NADH存在时,细胞色素b_5还原酶Ⅰ能够还原细胞色素b_5,其吸收峰从411 nm偏移至422 nm,并在521 nm和554 nm处吸光值增加。【结论】细胞色素b_5还原酶Ⅰ N端穿膜区域的去除增加了其可溶性,并保持了蛋白质活性;高山被孢霉ATCC 32222中细胞色素b_5还原酶Ⅰ基因编码的是一种NADH-细胞色素b_5还原酶,其在体外能与细胞色素b_5相互作用。     

NADH荧光法快速检测细菌总数

基于细菌胞内NADH的荧光特性及其在胞内含量稳定的特性, 建立一种快速检测细菌总数的新方法。该荧光法的NADH检测限为1 nmol/L, NADH含量在10 nmol/L~0.2 mmol/L间与荧光强度呈良好线性关系(R2 =0.9905)。经离心获得菌体细胞, 热Tris-HCl法提取胞内NADH, 以 342 nm为激发波长, 461 nm为发射波长测定提取液荧光强度, 1 h内可检测到样品1×104 CFU/mL菌数。结果表明该方法快速、灵敏、简便、重复性好, 可适用于食品卫生与安全、环境检测等领域活细菌数量的定量检测。     

转录调控因子Rex的功能及调节机制研究进展

转录因子Rex是一种广泛存在于革兰氏阳性菌,能够与NADH或者NAD⁺直接结合响应胞内NADH/NAD⁺的氧化还原传感器,与靶基因的结合可调节细胞内的多种生理代谢。NAD（H）是调节细胞能量代谢的必需辅酶,显示微生物细胞内的氧化还原状态。研究发现Rex的调节活性与细胞内NADH/NAD⁺比率相关。需氧和厌氧菌属中Rex单体和复合物晶体结构的解析揭示了Rex、NADH/NAD⁺和靶基因间的作用关系及调控机制。通过比较分析了不同菌株中Rex单体和复合物的晶体蛋白结构,并揭示了NADH/NAD⁺对Rex调控活性的影响,进一步解析了Rex与碳和能量代谢、厌氧代谢、发酵、生物膜等之间的联系,并展望了Rex的研究和应用方向。     

光对绿豆种子和叶片分离线粒体耗氧的影响

实验结果表明:照光时绿豆叶片分离线粒体通过细胞色素氧化酶途径的NADH氧化部分受阻,电子转向交替途径。不产生能量,不受能荷控制的NADH氧化途径有利于绿色细胞线粒体在光合作用时执行其提供碳架的功能。看来绿色细胞线粒体本身具有对光的敏感性,在照光时调节呼吸途径以适应其功能的转换。呼吸途径的转换机制目前还不清楚。绿豆种子线粒体与叶片线粒体不同,没有上述的这种对光的反应。     

NADH依赖型酶活性检测方法的建立

目的:建立一种NADH依赖型酶活性检测的方法。方法:将FDH、LeuDH串联克隆到表达载体pET-22b(+)中,转化至E.coli,并向培养液中分别添加去离子水、甲酸胺、三甲基丙酮酸,反应一段时间后,检测NADH的吸光度。同时通过测定氨气的产生判断FDH活性;通过薄层层析检测判断LeuDH活性;比较NADH吸光度测定结果与常规方法结果是否一致。结果:通过测定氨气的产生,证明FDH具有活性;此时NADH吸光度上升亦说明FDH具有活性;两种方法结论一致。薄层层析检测,生成叔亮氨酸,证明LeuDH具有活性;此时NADH吸光度下降亦说明LeuDH具有活性;两种方法结论一致。结论:通过检测菌体内部NADH吸光度的变化检测NADH依赖型酶活性的方法可行。     

胞内氧化还原水平对嗜热厌氧乙醇菌 发酵代谢的影响

辅酶NADH/NAD+在细胞内氧化还原反应中起着重要的作用,是细胞生长和能量代谢必不可少的辅因子。调节微生物胞内NADH/NAD+的比率是定向改变微生物代谢,高效获得目标代谢产物的有效手段。嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus)是高温厌氧菌中乙醇产量较高的代表性菌株,本文利用不同氧化还原态的碳源改变T.ethanolicus的胞内NADH/NAD+含量和比例,进而研究了其对细胞生长、代谢产物分布的影响。以不同比例的葡萄糖/甘露醇作为混合碳源发酵,胞内氧化还原水平、细胞的生长特性、代谢产物都发生了不同程度的差异,以葡萄糖作为唯一碳源进行培养时,T.ethanolicus生长良好,乙醇产量为0.79g/L,但胞内NADH/NAD+比值和乙醇/乙酸的比值都比较低,分别为0.47和4.82;随着葡萄糖在混合碳源中比例的下降,NADH/NAD+比值增高,发酵产物中乙醇/乙酸比值也呈现上升的趋势。而以甘露醇作为唯一碳源时,发酵产物中乙醇浓度为0.389g/L,NADH/NAD+比值和乙醇/乙酸的比值分别为1.04和16.0。     

光对绿豆种子和叶片分离线粒体耗氧的影响

实验结果表明：照光时绿豆叶片分离线粒体通过细胞色素氧化酶途径的NADH氧化部分受阻,电子转向交替途径。不产生能量．不受能荷控制的NADH氧化途径有利于绿色细胞线粒体在光合作用时执行其提供碳架的功能。看来绿色细胞线粒体本身具有对光的敏感性,在照光时调节呼吸途径以适应其功能的转换。呼吸途径的转换机制目前还不清楚。绿豆种子线粒体与叶片线粒体不同,没有上述的这种对光的反应。     

细菌荧光素酶体外发光体系的建立及应用于NADH定量检测

摘要：【目的】本研究旨在建立鳆发光杆菌（Photobacterium leiognathi）YL 荧光素酶：FMN-NADH氧化还原酶体外发光双酶体系,并对荧光素酶：FMN-NADH氧化还原酶体外发光双酶体系应用于NADH的定量检测进行初步探索。【方法】利用从鳆发光杆菌提取并经部分纯化的荧光素酶和FMN-NADH氧化还原酶,通过优化体系中各底物的添加量,实现荧光素酶的体外发光。【结果】荧光素酶：FMN-NADH氧化还原酶体外发光双酶体系为：1 mL酶液中添加100 μL十二烷醛（27 mmol/L）、0.5 μL FMN-Na（10 mmol/L）、300 μL NADH（0.14 mmol/L）。NADH与荧光素酶：FMN-NADH氧化还原酶体系的发光强度呈良好的线性关系,其线性范围为1.0×10-10 ～1.0×10-8 mol/L。【结论】荧光素酶：FMN-NADH氧化还原酶体外发光双酶体系可以简便、灵敏、快速的定量检测NADH,为其进一步应用于环境检测、食品卫生与安全等领域活细菌数量的检测奠定了基础。     

植物呼吸及代谢的研究 Ⅷ.水稻幼苗线粒体电子传递的多条途径

由差异光谱的测定结果表明,水稻幼苗的线粒体呼吸链存在细胞色素系统的所有电子载体:黄素蛋白、细胞色素 b、细胞色素 c、细胞色素 a 和细胞色素 a_3。用氧电极测定及铁氰化物还原的结果表明,在水稻线粒体中至少存在四条电子传递途径:第一条定位于线粒体内膜内侧,对鱼藤酮、抗霉素 A 及氰化物敏感;第二条也定位于线粒体内膜内侧,但对鱼藤酮不敏感;第三条能氧化外源的 NADH,对鱼藤酮不敏感,而受抗霉素 A抑制,其 NADH 脱氢酶定位于线粒体内膜外侧;第四条途径在氧化外源 NADH 时,如有外源细胞色素 c 存在,可通过外膜上对抗霉素 A 不敏感的 NADH 脱氢酶进行电子传递。由此可以得出结论,高等植物以水稻为代表,其线粒体中的电子传递途径也是多条的。     

牛磺酸的生物学效应与运动能力的研究进展

牛磺酸是人和动物的重要营养素,具有多种生物学作用,能清除自由基、对抗脂质过氧化、调节渗透压、维持体液平衡和胞内钙离子稳态、参与糖和氨基酸的代谢。在体育运动中,牛磺酸能显著提高运动能力。本文对牛磺酸在人体内的分布和代谢进行介绍,并对牛磺酸在人体内的功能及其与运动能力的关系进行概述。     

氢气与肠道菌群的关系研究进展

目前,氢气已被证实在多种疾病中具有显著的医学效应,然而其发挥效应的分子机制并不清楚。肠道菌群被人们看作人体的一个重要“器官”,与人类健康的关系密不可分。研究表明,人类肠道菌群中存在着大量能够进行氢气代谢的菌群,这些菌群的变化可能与多种疾病的发生发展密切相关。此外,研究还发现外源氢气干预可能通过重塑肠道菌群改善炎症性肠病、脂肪性肝病等。综述了肠道菌群的氢气代谢及其与疾病发生发展的关系以及外源氢气干预通过调节肠道菌群影响疾病进展的相关研究,希望能为致力于从肠道菌群角度研究氢气医学效应的科研工作者提供帮助。     

Potential use of radiation diagnosis of esophageal achalasia

The prime objective of the paper is to attempt to reevaluate the potentialities of current radiation diagnosis of esophageal achalasia. Moreover, its main idea is to assess the procedure of a traditional X-ray section of radiation study. In the authors' opinion, the main motivation of writing this paper was the emergence of new recent potentialities of radiation diagnosis, including its traditional X-ray section in gastroenterological pathology, as well as a considerable body of information on esophageal achalasia (144 cases). The tasks to be solved in the paper include the differential diagnosis of esophageal achalasia and cardioesophageal cancer, as well as the basic capacities of radiation diagnosis to help clinicians in characterizing the changes caused by medical and surgical treatments for this disease. The authors consider that the obtained volume of necessary information on this abnormality rather frequently encountered in esophagogastroenterological pathology can be increased by applying the current possibilities of radiation study and mainly its traditional X-ray section.     

A Translatable Predictor of Human Radiation Exposure

Terrorism using radiological dirty bombs or improvised nuclear devices is recognized as a major threat to both public health and national security. In the event of a radiological or nuclear disaster, rapid and accurate biodosimetry of thousands of potentially affected individuals will be essential for effective medical management to occur. Currently, health care providers lack an accurate, high-throughput biodosimetric assay which is suitable for the triage of large numbers of radiation injury victims. Here, we describe the development of a biodosimetric assay based on the analysis of irradiated mice, ex vivo-irradiated human peripheral blood (PB) and humans treated with total body irradiation (TBI). Interestingly, a gene expression profile developed via analysis of murine PB radiation response alone was inaccurate in predicting human radiation injury. In contrast, generation of a gene expression profile which incorporated data from ex vivo irradiated human PB and human TBI patients yielded an 18-gene radiation classifier which was highly accurate at predicting human radiation status and discriminating medically relevant radiation dose levels in human samples. Although the patient population was relatively small, the accuracy of this classifier in discriminating radiation dose levels in human TBI patients was not substantially confounded by gender, diagnosis or prior exposure to chemotherapy. We have further incorporated genes from this human radiation signature into a rapid and high-throughput chemical ligation-dependent probe amplification assay (CLPA) which was able to discriminate radiation dose levels in a pilot study of ex vivo irradiated human blood and samples from human TBI patients. Our results illustrate the potential for translation of a human genetic signature for the diagnosis of human radiation exposure and suggest the basis for further testing of CLPA as a candidate biodosimetric assay.     

Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies

Normal mitochondrial function is a critical factor in maintaining cellular homeostasis in various organs of the body. Due to the involvement of mitochondrial dysfunction in many pathological states, the real-time in vivo monitoring of the mitochondrial metabolic state is crucially important. This type of monitoring in animal models as well as in patients provides real-time data that can help interpret experimental results or optimize patient treatment. The goals of the present review are the following: 1) to provide an historical overview of NADH fluorescence monitoring and its physiological significance; 2) to present the solid scientific ground underlying NADH fluorescence measurements based on published materials; 3) to provide the reader with basic information on the methodologies used in the past and the current state of the art fluorometers; and 4) to clarify the various factors affecting monitored signals, including artifacts. The large numbers of publications by different groups testify to the valuable information gathered in various experimental conditions. The monitoring of NADH levels in the tissue provides the most important information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels. Although NADH signals are not calibrated in absolute units, their trend monitoring is important for the interpretation of physiological or pathological situations. To understand tissue function better, the multiparametric approach has been developed where NADH serves as the key parameter. The development of new light sources in UV and visible spectra has led to the development of small compact units applicable in clinical conditions for better diagnosis of patients. real-time tissue viability; tissue spectroscopy; patient monitoring     

MicroRNAs: Crucial multi-tasking components in the complex circuitry of tumor metastasis

Distant metastases are the underlying cause of patient mortality in an overwhelming majority of human carcinomas. Certain microRNAs have recently been found capable of regulating the process of tumor metastasis. In this review, we highlight recent advances within this rapidly emerging field, endeavor to connect known microRNA pathways with recent conceptual advances in the larger field of metastasis research, and speculate regarding the future utility of microRNAs in the diagnosis and treatment of human cancers. Assessed collectively, current evidence suggests that the pleiotropic activities of microRNAs endow them with the capacity to function as crucial, yet previously unappreciated, nodes within already-identified metastasis regulatory circuitry. This has important implications for our understanding of the pathogenesis of high-grade malignancies.     

Regulatory initiatives for natural latex allergy: US perspectives

The US Food and Drug Administration (FDA) has regulatory authority over foods, human drugs, cosmetics, medical devices, radiological products, biologics, and veterinary products. Among these products, FDA believes that the use of medical devices, including medical gloves, condoms, catheters, and breathing bags, represents the greatest source of natural latex proteins to exposed individuals. A medical device is defined in the Federal Food Drug and Cosmetic Act (FFDCA) as an instrument, apparatus, implement, machine, etc., that is intended for use in the diagnosis or treatment of disease or is intended to affect the structure or any function of the body of a human or other animal, and that does not achieve any of its principal intended purposes through chemical action in the body. This article provides some brief, general background about FDA's medical device regulatory process and then addresses the issue of natural latex allergy. Finally we discuss the steps the Agency has taken to evaluate the magnitude and nature of the problem, and FDA's efforts to assist manufacturers, health professionals, and others in minimizing exposure and sensitization to natural latex proteins in medical devices.     

Noninvasive techniques for measuring body elemental composition

In the past 20 yr, in vivo analysis of body elements by neutron activation has become an important tool in medical research. In particular, it provides a much needed means to make quantitative assessments of body composition of human beings in vivo. The data are useful both for basic physiological understanding and for diagnosis and management of a variety of diseases and disorders. This paper traces the development of the in vivo neutron activation technique from basic systems to the present state of the art facilities. A scan of some of the numerous clinical applications that have been made with this technique reveals the broad potentialities of in vivo neutron activation. The paper also considers alternative routes of future development and raises some of the questions now faced in making the technique more widely available to both medical practitioners and medical investigtors. In vivo neutron activation has opened a new era of both clinical diagnosis and therapy evaluation, and investigation into the modeling of body composition. The techniques are new, but it is already clear that considerable strides can be made in increasing accuracy and precision, increasing the number of elements susceptible to measurement, and reducing the dose required for the measurement.     

普鲁士蓝纳米粒子在生物医学诊断和治疗中的应用及进展

随着生物医学诊断和治疗的持续深入研究,出现了多种医学诊断和治疗新方法,为人类的健康提供了更大的保证,其中纳米生物技术在生物医学诊断和治疗中的应用日益增多,基于纳米技术,开发传统材料的生物医学新应用成为了人们的研究热点。普鲁士蓝是一种历史悠久的蓝色染料,其制备过程简单、绿色、成本低,化学结构稳定,具有优良的物理、化学、光学以及磁性等性能,已经在许多领域得到了广泛的应用。近年来,普鲁士蓝开始在生物医学诊断和治疗领域中崭露头角,它已经成功的被开发为新型的核磁共振造影剂和光声成像造影剂,并且在药物输送系统和光热治疗等领域也开始占有一席之地,开发基于纳米技术的普鲁士蓝的生物医学应用已经成为极具吸引力的研究方向。本文对普鲁士蓝在生物医学诊断和治疗中的应用及进展进行综述。     

Lessons from Queer Bioethics: A Response to Timothy F. Murphy

‘Bioethics still has important work to do in helping to secure status equality for LGBT people’ writes Timothy F. Murphy in a recent Bioethics editorial. The focus of his piece, however, is much narrower than human rights, medical care for LGBT people, or ending the HIV/AIDS pandemic. Rather, he is primarily concerned with sexuality and gender identity, and the medical intersections thereof (i.e. DSM diagnosis; access to SrS or ARTs). It is the objective of this response to provide an alternate account of bioethics from a Queer perspective. I will situate Queer bioethics within Queer studies, and offer three ‘lessons’ that bioethics can derive from this perspective. These are not definitive rules for Queer bioethics, since it is a field which fundamentally opposes categorizations, favoring pastiche over principles. These lessons are exploratory examples, which both complement and contradict LGBT bioethics. My latter two lessons – on environmental bioethics and disability – overlap with some of Murphy's concerns, as well as other conceptions of LGBT bioethics. However, the first lesson takes an antithetical stance to Murphy's primary focus by resisting all forms of heteroconformity and disavowing reproduction as consonant with Queer objectives and theory. The first lesson, which doubles as a primer in Queer theory, does heavy philosophical lifting for the remainder of the essay. This response to Timothy F. Murphy, whose work is certainly a legacy in bioethics, reveals the multiplicity of discourses in LGBT/Queer studies, many of which are advantageous – even essential – to other disciplines like bioethics.