激光诱导荧光光谱中内源性原卟啉Ⅸ对胃癌生长状态的标示作用

用不同的生物治疗方式治疗人胃癌的裸鼠模型,通过测量治疗后的瘤体体积和检查病理组织学的变化,确定了各组胃癌组织中癌细胞的不同坏死程度。应用激光诱导自体荧光光谱系统测量不同坏死程度的胃癌组织的自体荧光光谱。结果发现,随肿瘤坏死程度的加重,自体荧光光谱中内源性原卟啉Ⅸ(Pp Ⅸ)的特征峰值降低,而烟酰胺腺嘌呤二核苷酸(NADH)的特征峰未能提示该变化。表明Pp Ⅸ可以作为标识,灵敏地表征胃癌组织的生长状态。     

激光诱导荧光光谱中内源性原卟啉IX对胃癌生长状态的标示作用

用不同的生物治疗方式治疗人胃癌的裸鼠模型,通过测量治疗后的瘤体体积和检查病理组织学的变化,确定了各组胃癌组织中癌细胞的不同坏死程度.应用激光诱导自体荧光光谱系统测量不同坏死程度的胃癌组织的自体荧光光谱.结果发现,随肿瘤坏死程度的加重,自体荧光光谱中内源性原卟啉Ⅸ(Pp Ⅸ)的特征峰值降低,而烟酰胺腺嘌呤二核苷酸(NADH)的特征峰未能提示该变化.表明Pp Ⅸ可以作为标识,灵敏地表征胃癌组织的生长状态.     

低强度激光血管内照射治疗的机理初探

用532nm的激光作为激光光源分别测量正常血液、血浆及HBsAg阳性血液、血浆的荧光光谱。血液光谱的测量结果显示,两种血液樯本都在630nm及710nm附近出现有荧光峰值。从光谱学的角度分析,可得出激光照射血液治疗的内在机制在激光照射可增强血液的携氧能力,从而促进了机体的新陈代谢。血浆图谱测量结果显示,正常血浆及HBsAg阳性血浆在738nm处的荧光强度有显著差异,可作为临床光谱诊断中判断HBsAg阳性的标准和依据。对HBsAg阳性血液的生化检测结果显示,He-Ne激光照射可抑制或杀伤乙肝病毒。     

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。     

NADH-硝酸还原酶组分酶的活性测定

NADH—硝酸还原酶(NADH—nitrate reduc—tase,EC 1.6.6.1,NADH—NR)是硝态氮同化的关键酶,它能以NADH为电子供体,还原NO_3~-为NO_2~-。由于它在植物氮代谢中的重要作用,国内外已对它的诱导和活性调节进行了广泛的研究。 NADH—NR是组分酶复合物,改变电子供体或受体,可测到 NADH—NR的二个组分酶活性,     

细菌nox基因研究进展

细菌nox基因编码合成一种含核黄素的NADH氧化酶,NADH氧化酶可催化双原子氧还原为H2O2或H2O,同时将NADH氧化为NAD+。该反应发生在多种代谢途径中,从而对细菌的氧化应激、菌膜形成、毒力调控及代谢产物生成等生理生化过程产生一系列影响。目前对高等动植物体中的nox基因及其编码的NADH氧化酶已有较深入的研究,但近年来一些研究表明,细菌nox基因的功能及作用通路与动植物体存在较大差异,因此,有必要详细了解细菌中nox基因和NADH氧化酶的具体作用机制及其对细胞产生的影响。综合分析近年来细菌nox基因及NADH氧化酶的研究成果,结合我们的研究,对目前存在的问题和未来的发展进行综述。     

猪肝二氢蝶啶还原酶的提取及动力学研究

本文报导了从猪肝中提取二氢蝶啶还原酶[Ecl.6.99.7]的方法,提取百分率达30％左右。以DMPH_4为底物,分别以NADH和NADPH为辅酶,测定了该酶的动力学,发现它对NADH具有一定的特异性[Km(NADH)Vmax(NADPH)]。不同的金属离子对该酶活性影响的程度有很大的差异。     

肿瘤的荧光诊断

利用荧光技术对恶性肿瘤进行诊断 ,至今仍是人们较为关注的一个课题。本文对肿瘤荧光诊断的现状进行了评述。肿瘤荧光诊断的最基本的依据是 :恶性肿瘤患者其肿瘤组织或体液的新陈代谢将发生异常 ,通过对这些异常物质的荧光检测 ,有望诊断肿瘤。肿瘤荧光诊断现有两种观点 :1 建立在卟啉代谢异常的基础上实验依据是 ,恶性肿瘤组织或患者的体液 (如血清 ) ,除正常荧光峰外 ,在 6 30 μｍ或 6 70 μｍ附近有一附加荧光峰 ,称之为肿瘤的“特征峰”或“固有荧光”。这附加的荧光峰和卟啉的荧光类似。认为这种附加峰是患者出现卟啉代谢异常的结果…     

毕赤酵母木糖还原酶定点突变改善其对双辅酶的亲和力

通过毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase, XR)基因定点突变,获得NADH高亲和力的毕赤酵母木糖还原酶(PsXR),改善了辅酶不同而导致的酿酒酵母胞内氧化还原失衡.同时克隆了PsXR编码基因,通过BLAST工具进行同源性搜索,并用生物软件进行序列比对和结构分析,确定突变位点.用融合PCR方法进行定点突变,并在大肠杆菌表达系统中进行融合表达,且对表达产物进行HIS-TAG亲和纯化,分光光度法检测酶活性,计算比活力.本研究成功获得突变XR编码基因,并收集了纯化的突变蛋白.酶活性检测和比活力计算显示,3种突变酶对2种辅酶的亲和力在一定程度上都发生了变化.与未突变的PsXR相比,3种突变酶对辅酶NADPH的亲和力均显著下降,突变酶M3对辅酶NADH的亲和力未发生变化,而突变酶M1和M4对辅酶NADH的亲和力显著升高,其中突变酶M1对NADH的亲和力明显提高,对NADPH的亲和力明显下降,其活性主要依赖辅酶NADH,提示K270R位点在XR与辅酶结合中起关键作用.     

高盐下条斑紫菜光合特性和S-腺苷甲硫氨酸合成酶基因表达的变化

为了研究条斑紫菜耐盐机理,对条斑紫菜叶状体进行了高盐胁迫处理,继而采用氧电极法测量了光合放氧速率和呼吸耗氧速率的变化,采用实时荧光定量PCR技术测量了S-腺苷甲硫氨酸合成酶(命名为PySAMS)基因的表达变化。结果显示藻体的光合与呼吸作用均受到高盐度海水的显著影响,随着盐度的增加,光合放氧率逐渐降低,呼吸耗氧率也逐渐降低。高盐度海水对PySAMS基因表达量也产生了显著影响,40和50盐度的海水诱导了PySAMS表达,但60至80盐度的海水却不同程度地抑制了PySAMS表达。据此推测,在面对较高盐度胁迫时条斑紫菜叶状体将逐步降低体内新陈代谢以度过不良环境。     

The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions

The reduced coenzyme NADH plays a central role in mitochondrial respiratory metabolism. However, reports on the amount of free NADH in mitochondria are sparse and contradictory. We first determined the emission spectrum of NADH bound to proteins using isothermal titration calorimetry combined with fluorescence spectroscopy. The NADH content of actively respiring mitochondria (from potato tubers [Solanum tuberosum cv Bintje]) in different metabolic states was then measured by spectral decomposition analysis of fluorescence emission spectra. Most of the mitochondrial NADH is bound to proteins, and the amount is low in state 3 (substrate + ADP present) and high in state 2 (only substrate present) and state 4 (substrate + ATP). By contrast, the amount of free NADH is low but relatively constant, even increasing a little in state 3. Using modeling, we show that these results can be explained by a 2.5- to 3-fold weaker average binding of NADH to mitochondrial protein in state 3 compared with state 4. This indicates that there is a specific mechanism for free NADH homeostasis and that the concentration of free NADH in the mitochondrial matrix per se does not play a regulatory role in mitochondrial metabolism. These findings have far-reaching consequences for the interpretation of cellular metabolism.     

Altered NADH and improved function by anesthetic and ischemic preconditioning in guinea pig intact hearts

NADH increases during ischemia because O(2) shortage limits NADH oxidation at the electron transport chain. Ischemic (IPC) and anesthetic preconditioning (APC) attenuate cardiac reperfusion injury. We examined whether IPC and APC similarly alter NADH, i.e., mitochondrial metabolism. NADH fluorescence was measured at the left ventricular wall of 40 Langendorff-prepared guinea pig hearts. IPC was achieved by two 5-min periods of ischemia and APC by exposure to 0.5 or 1.3 mM sevoflurane for 15 min, each ending 30 min before 30 min of global ischemia. During ischemia, NADH initially increased in nonpreconditioned control hearts and then gradually declined below baseline levels. This increase in NADH was lower after APC but not after IPC. The subsequent decline was slower after IPC and APC. On reperfusion, NADH was less decreased after IPC or APC, mechanical and metabolic functions were improved, and infarct size was lower compared with controls. Our results indicate that IPC and APC cause distinctive changes in mitochondrial metabolism during ischemia and thus lead to improved function and tissue viability on reperfusion.     

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD⁺]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD⁺]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD⁺]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD⁺]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD⁺]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.     

Determination of NADH in frozen rat brain sections by laser-induced fluorescence

Methods to assess metabolism are important analytical tools in neuroscience. The fluorophore nicotinamide adenine dinucleotide (NADH) is a parameter of cellular metabolism. NADH fluorescence was measured using a laser-based fluorescence detector with spectral and temporal filters. Distribution and intensity of NADH fluorescence were investigated in frozen brain sections. In sections containing hippocampus the intensity of NADH fluorescence was correlated to brain structures. In order to investigate the consequences of neurotoxic lesions, 5,7-dihydroxytryptamine was injected into the dorsal raphe nucleus 4 to 240 days prior to the measurement. NADH fluorescence decreased in the affected region by 50%, indicating that no recovery in metabolic activity had occurred.     

Genetically encoded fluorescent sensors for intracellular NADH detection

We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.     

Multiple proteins with single activities or a single protein with multiple activities: the conundrum of cell surface NADH oxidoreductases

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.     

Electrical stimulation of the energy metabolism in yeast cells using a planar Ti-Au-Electrode interface

We report on the influence of dielectric pulse injection on the energy metabolism of yeast cells with a planar interdigitated electrode interface. The energy metabolism was measured via NADH fluorescence. The application of dielectric pulses results in a distinct decrease of the fluorescence, indicating a response of the energy metabolism of the yeast cells. The reduction of the NADH signal significantly depends on the pulse parameters, i.e., amplitude and width. Furthermore, the interface is used to detect electrical changes in the cell-electrolyte system, arising from glucose-induced oscillations in yeast cells and yeast extract, by dielectric spectroscopy at 10 kHz. These dielectric investigations revealed a β₁-dispersion for the system electrolyte/yeast cells as well as for the system electrolyte/yeast extract. In agreement with control measurements we obtained a glycolytic period of 45s for yeast cells and of 11min for yeast extract.     

The relationship between respiration rate and peroxidase activities in maize root mitochondria

In the present study we provide the evidence of different respiration rates and peroxidase activities in maize (Zea mays L.) mitochondria isolated from germinated seeds and roots of 2-week-old seedlings. The negative relationships between mitochondrial respiration rate measured with NADH as substrate and activities of peroxidases that oxidized NADH in both oxidative and peroxidative cycles were found. The possible role of peroxidase in the regulation of reactive oxygen species metabolism in expense of NADH oxidation was hypothesized.     

Nitric Oxide in Plants: The Roles of Ascorbate and Hemoglobin

Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia.