Measurement of EROD Activity: Caution on Spectral Properties of Standards Used

The activity of the enzyme 7-ethoxy-resorufin-O-deethylase (EROD) has been extensively used in biomonitoring studies for more than a decade. Although the analytical procedure is simple, it is often poorly characterized. In this study spectral properties of particular standard compounds used to measure EROD activity (ethoxyresorufin and resorufin, standards from Molecular Probes) were tested in order to optimize excitation and emission wavelengths to be used in the fluorimetric assay of EROD activity. The optimal excitation wavelength for the detection of resorufin was 560 nm. At this wavelength the excitation represents only 37% of its maximum level for ethoxyresorufin, while it represents 86% for resorufin. This allows discrimination between the fluorescence emitted by both standards, favoring the formed resorufin. Our results demonstrate that any analytical work using spectrofluorometry to measure EROD activity should be preceded by precise determination of the spectral characteristics of each set of standards used.     

Evidence for the Uptake of Large Anions through Stomatal Pores

Experiments were conducted with leek (Allium porrum L.) leaves to investigate whether aqueous solutions are able to penetrate stomata. Epidermal strips were used for the determination of transport rates. Stomata were opened by fusicoccin or closed by darkness or abscisic acid. A droplet containing the anionic fluorescent dye, uranine, was placed on the physiologically outer side of the epidermis and allowed to dry. With open stomata 30 times more uranine penetrated through the epidermal strips than with closed stomata (comparison of medians). In another experiment droplets of uranine solution were placed on leaf segments and epidermal strips were removed after drying of the droplets. Penetration of uranine through stomata was detectable under the microscope both with epidermal strips from the transport experiments and with strips obtained after application on leaf segments. As maximum uptake rates occurred during the drying process, it is concluded that penetration took place via water films. These results show that the physical restrictions preventing stomatal penetration of static droplets are not decisive for drying droplets and that stomatal uptake of dissolved ionic substances occurs under natural conditions, i.e. without surfactants or applied pressure.     

On-line process monitoring and chemometric modeling with 2D fluorescence spectra obtained in recombinant E. coli fermentations

2D spectrofluorometry produces a large volume of spectral data during fermentation processes with recombinant E. coli, which can be analyzed using chemometric methods such as principal component analysis (PCA), principal component regression (PCR) and partial least square regression (PLS). An analysis of the spectral data by PCA results in scores and loadings that are not only visualized in the score-loading plots but are also used to monitor the fermentation processes on-line. The score plots provided useful qualitative information on four fermentation processes for the production of extracellular 5-aminolevulinic acid (ALA). Two chemometric models (PCR and PLS) were used to examine the correlation between the 2D fluorescence spectra and a few parameters of the fermentation processes. The results showed that PLS had slightly better calibration and prediction performance than PCR.     

Attachment of A172 human glioblastoma cells affects calcium signalling: a comparison of image cytometry, flow cytometry, and spectrofluorometry.

The intracellular free calcium concentration ([Ca2+]i) of indo-1 loaded A172 human glioblastoma cells stimulated by platelet-derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry. With all three techniques, the percentage of responsive cells, peak [Ca2+]i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose. The maximum response occurred at a PDGF concentration of about 20 ng/ml. Basal and peak [Ca2+]i did not differ significantly from method to method even though different calibration procedures were used. Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells. This did not appear to be a direct effect of trypsinization. Spectral analysis of indo-1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency. Calibration of [Ca2+]i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.     

Spatial distributions of foliar nitrogen and phosphorus in crowns of Eucalyptus grandis

Summary Eucalyptus grandis trees were grown in plantations with and without added fertiliser to examine the effects of plant nutrition on photosynthesis and growth. Leaves were sampled from known locations within canopies of selected trees and leaf N and P concentrations were measured. Contour maps of N and P distributions were then produced for crowns of trees aged between 6 and 16 months. Gas exchange measurements on sample leaves were used to estimate parameters of a model of C₃ photosynthesis as a function of leaf N and P contentrations. Linear relationships were obtained between model parameters and leaf N concentration, but P appeared to be present in excess, since no correlation was found with P contentration. Photosynthetic light response curves were calculated for model leaves with differing N concentrations. The curves show that optimal concentrations of N in leaves depend on mean levels of irradiance during growth.     

Ethylene-Enhanced Transport of Uranine, a Fluorescent Dye, in Rice-Seedling Explants in Relation to Ethylene-Stimulated Coleoptile Growth

Ethylene enhanced the transport of uranine, a fluorescent dye,through the coleoptiles of rice (Oryza sativa L. cv. Sasanishiki)seedlings explants after a 2-h lag period. The uranine transportincreased with increasing concentration of ethylene, reachinga maximum at 1 µ1.liter^–1 ethylene. It decreasedwhen some metabolic inhibitors, 2,4-dinitrophenol, carbonylcyanide 3-chlorophenylhydrazone and N-ethylmaleimide, were appliedto the scutella. Mechanical rupture or heating at the vascular-bundlesides of the coleoptiles also interfered with the ethylene-enhancedtransport of uranine. The wounding and removal of the scutellasimilarly blocked uranine transport. Moreover, strong fluorescenceof uranine was observed on the phloem regions of two vascularbundles of a coleoptile under a fluorescence microscope. Itis suggested that the ethylene-enhanced transport of uraninein the coleoptile of rice-seedling explants occurs through thephloem. On the other hand, the removal of the endosperm or therupture of the vascular bundles inhibited the ethylene-stimulatedelongation of the rice coleoptiles. Moreover, ethylene increaseddry weight of the shoots. These results suggest that the ethylene-stimulatedelongation of rice coleoptiles was supported partly by the ethylene-enhancedtranslocation of materials from the endosperms to the coleoptiles. (Received September 6, 1984; Accepted November 15, 1984)     

Phase Changes in Amylose–Butanol Complex Solution

The phase change for an amylose–butanol complex solution in 10% of dimethylsulfoxide was investigated as a function of temperature. The phase change was determined with measurements of the turbidity, fluorescent depolarization, and viscosity. The phase diagram obtained was qualitatively similar to that for an amylose solution. From the result, the change in solution phase for the amylose–butanol complex is suggested to be similar to that for amylose, i.e., when the solution cools from a higher temperature, amylose molecules in the complex solution change the conformation from a random coil to an interrupted helix, and then separate into two phases. Coacervate particles resulting from the phase separation coalesce with each other to yield precipitates.

An adsorption of uranine on amylose was studied to ascertain its relationship with the fluorescent depolarization method used for detecting phase changes in solution. The result showed that uranine was adsorbed on amylose chains but not on the amylose–butanol complex.     

基于小波理论的干旱区内陆湖泊叶绿素a的TM影像遥感反演

叶绿素a(Chl-a)是衡量湖泊富营养化的重要指标,利用遥感技术动态监测面积较大的湖区水体中Chl-a浓度对了解湖区水质具有重要意义。以内蒙古乌梁素海为例,提出利用TM影像中的水体实测光谱进行小波去噪和光谱信号重构,并结合水质采样实测数据进行神经网络拟合,建立光谱反射率比值与Chl-a浓度的反演模型的方法。结果显示:小波理论和神经网络相结合的模型可以适用于估算乌梁素海Chl-a浓度,去噪后Chl-a浓度与光谱信号的相关系数(-0.575)较去噪前(-0.417)明显增强,去噪后的采样点光谱信号与Chl-a浓度之间表现出比原始信号更强的负相关性,证明了去噪后的观测值可进一步减弱随机误差的干扰和去除噪声,使观测数据更加逼近Chl-a浓度的真实情况,图像去噪重构结果显示重构后的光谱范围较之前有所缩窄,部分信号点得到了增强,但基本剖面结构并没有产生较大变化,反演模型的平均相对误差为0.142,与其他研究相比差别不大。反演得出的乌梁素海Chl-a浓度分布反映了污染源的分布,同时说明了乌梁素海Chl-a浓度在时空分布上呈现一定的差异,表现为丰水期呈现浅水区Chl-a浓度值高于湖心区,来水区高于其他湖区的分布趋势,枯水期乌梁素海中部呈现由西向东Chl-a浓度逐步降低的分布规律,西部呈均一化分布。反演模型基本可以满足实际预测的需要。但模型在具体应用中在影像数据采集、数据量及算法方面还有很大的改进空间,该方法的提出为干旱区大型内陆水体富营养化的实时定量遥感监测提供了新的解决方案。     

Sources of uncertainty in assessment of marine phytoplankton communities

Characterisation of phytoplankton communities is important for classification of the ecological status of marine waters. In order to design a monitoring programme, it is important to know what degree of variation in the measurements occur at each level (water body, station and sample), so that resources can be spent in a way that maximise the precision of the measured parameters. Seven European water bodies were sampled to assess the variation in pigment concentrations and population densities attributed to water body, station and sample levels. It was found that the main proportion of the variation between pigment measurements was explained by the variation between stations (12–91% of variation) followed by the variation between water bodies (0–89% of variation). For measurements of population density, the main proportion of the variation between densities of cells recorded was explained by the variation between the taxonomists counting the samples (61%), whilst the main proportion of the variation between numbers of taxa recorded was explained by the variation between water bodies (83%). When the cell density of the nine dominant classes were analysed separately, the main proportion of variation was explained at the water body level for all but two class.     

CD of nucleic acids: III. Calculated CD of RNAs from new A, U, G, and C transition-moment parameters

New adenine (A) and uracil (U) π → π* transition-moment parameters have been derived from a recently developed semiempirical procedure. Using conformational energy probabilities based on the Boltzmann equation, the new parameters were assigned by optimizing the calculated CD of cyclic nucleotides against measured CD. The derived A-and U-parameters (along with guanine and cytosine parameters derived previously by the same procedure) have been assessed in CD spectral calculations of some polyribonucleic acid sequences, in assumed A-class geometries. Comparisons have been made between CD spectra calculated from the newly derived parameters and those calculated from parameters obtained from a combination of crystal optical measurements and quantum-mechanical calculations. Although some spectral differences do occur, for the RNA sequences considered, no major disagreements were found in CD spectral signs and shapes, between measurements and calculations. Overall, the results indicate that the newly derived A-, U-, G-, and C-parameters show better agreement between theory and experiment than those used in previous nucleic acid CD calculations.     

Relaxation of water protons in highly concentrated aqueous protein systems studied by 1H NMR spectroscopy

Concentrated Aqueous Protein Systems, Proton Relaxation Times, Slow Chemical Exchange In this paper we present proton spin-lattice (T1) and spin-spin (T2) relaxation times measured vs. concentration, temperature, pulse interval (tauCPMG) as well as 1H NMR spectral measurements in a wide range of concentrations of bovine serum albumin (BSA) solutions. The anomalous relaxation behaviour of the water protons, similar to that observed in mammalian lenses, was found in the two most concentrated solutions (44% and 46%). The functional dependence of the spin-spin relaxation time vs. tauCPMG pulse interval and the values of the motional activation parameters obtained from the temperature dependencies of spin-lattice relaxation times suggest that the water molecule mobility is reduced in these systems. The slow exchange process on the T2 time scale is proposed to explain the obtained data. The proton spectral measurements support the hypothesis of a slow exchange mechanism in the highest concentrated solutions. From the analysis of the shape of the proton spectra the mean exchange times between bound and bulk water proton groups (tauex) have been estimated for the range of the highest concentrations (30%-46%). The obtained values are of the order of milliseconds assuring that the slow exchange condition is fulfilled in the most concentrated samples.     

干旱胁迫下雷竹叶片叶绿素的高光谱响应特征及含量估算

植物叶片的反射光谱特征与叶绿素含量密切相关。以重要的笋用竹种雷竹(Phyllostachys violascens)为研究对象,采用盆栽及控水试验方法研究了2年生雷竹在干旱胁迫条件下冠层叶片反射光谱的响应特征,分析了叶片叶绿素含量与不同波段光谱反射率一阶微分值以及光谱特征参数之间的相关关系,并以雷竹叶绿素含量敏感波段及构建的植被指数与叶绿素含量进行了拟合。结果表明,重度缺水处理后雷竹叶片叶绿素含量显著降低,在可见光区叶片光谱反射率随叶绿素含量的降低而增加,以波长493、639、693、756 nm等处的光谱反射率一阶微分值与叶绿素含量的相关性较高。雷竹叶片叶绿素含量与光谱特征参数如绿峰反射率、红谷反射率、蓝边面积、绿峰面积之间的相关性较高。与已有的植被指数相比基于雷竹叶绿素含量敏感波段修正后的植被指数与叶绿素含量相关性优于原植被指数。基于反射率一阶微分值构建的多元回归方程以及修正的绿色归一化植被指数(m GNDVI)构建的回归方程拟合效果较好,为雷竹叶绿素含量的较优估算方程。研究结果可以为雷竹叶绿素含量的快速无损测定以及季节性干旱条件下雷竹林的科学经营及灾后评估提供依据。     

Effect of biomass adaptation to biodegradation of dissolved organic carbon in water

In the present study the time of adaptation of fixed biomass for biodegradation of natural organic matter was investigated. The experiments were done in columns that are usually used for rapid determination of biodegradable dissolved organic carbon (BDOC). The biomass was adapted to samples with different concentrations of organic substances before measurements by pumping water to be investigated through the columns for several days. The time of adaptation was dependent on the initial concentration of the organic matter in the water sample. The adaptation time increased from 6 to 24 h with increase of concentration of acetate solution from 2 to 10 mg/l, thus adaptation rate decreased simultaneously from 0.28 to 0.11 min⁻¹. In natural water samples with the initial concentration in the range from 4.61–10.82 mg/l of dissolved organic carbon (DOC) the maximal adaptation time was less than 24 h. During the adaptation period the increase in reproducibility and decrease in the standard deviation was observed. The study showed that adaptation of column to the different concentration of organic matter in water sample is necessary in order to decrease the bias in BDOC measurements when using columns tests.     

Reflectance and transmittance spectra of leaves and shoots of 22 vascular plant species and reflectance spectra of trunks and branches of 12 tree species in Japan

This data paper reports spectral reflectance and transmittance data of leaves from 21 terrestrial vascular plant species (seven herbaceous, and 14 broadleaf and long-needle coniferous tree species) and of shoots from one short-needle coniferous tree species. The reflectance spectra of branches of one tree species, of the trunks of 12 tree species and ground surface of one deciduous broad-leaf forest are also reported. Optical measurements and leaf samplings were made at five sites on Honshu Island, Japan, which are typical vegetation types in East Asia, i.e., grassland, paddy field, and deciduous broad-leaf or coniferous forests. The collection and measurements were conducted for main species in each site. To include other common vegetation types in East Asia, such as evergreen broad-leaf or coniferous forests, the sample collection and the measurements were conducted at gardens and an experimental forest. Leaves of ten deciduous species were measured at different phenological stages from leaf expansion to senescence since those species shows significant seasonal changes in spectral reflectance and transmittance of leaves. Leaves at different position in a canopy (e.g., sunlit versus shaded leaves) were also measured for eight of 21 species. The spectral reflectance and transmittance from both adaxial and abaxial sides of the all leaves or needles, expect Picea abies needles. The measurements of the leaves were conducted with a spectroradiometer attached via an optical fiber to an integrating sphere. Two types of integrating spheres were used: a model LI-1800-12 (Li-Cor) and an RTS-3ZC integrating sphere (Analytical Spectral Devices). A leaf clip accessory was also used instead of an integrating sphere for measuring the leaves of two species. All data were measured within the 350–2,500-nm spectral range with 1-nm steps between measurements but the data obtained by LI-1800 is unavailable in 1,650–1,740, 1,890–1,950, and 2,050–2,500 nm because of a large amount of noise. These data are used as input parameters in a radiative transfer model designed to estimate the leaf area index from radiation reflected from a canopy surface.     

水体悬浮物浓度对大型沉水植物黑藻反射光谱的影响

沉水植物的恢复是改善富营养化水体和重建水生生态系统的有效措施．应用遥感技术可以实时、大面积监测沉水植物的分布和生长情况,而水体中的悬浮物等物质影响沉水植物的光谱特征,直接影响对湖泊、河流遥感影像中沉水植物的准确解译．本研究利用地物光谱仪在室外实测了不同水体悬浮物浓度条件下沉水植物黑藻群落的光谱反射率．结果表明,在400～900nm波段,水体中悬浮物浓度越高,沉水植物的光谱反射率越大．水体悬浮物浓度与黑藻群落光谱反射率在400～900nm均为显著相关（P〈0．05）,在450～900nm为极显著相关（P〈0．01）．在蓝、绿、红和近红外4个波段,将水体悬浮物浓度与其光谱反射率进行回归分析,均得到了较好的线性关系．利用本研究结果对富含悬浮物水体中沉水植物的反射光谱进行修正,可为准确、快速和大尺度遥感监测沉水植物的分布和动态提供科学依据．     

An EPR study of the interfacial properties of phosphatidylcholine vesicles with different lipid chain lengths

The hydrophobic spin probe 2,2,6,6-tetramethyl-piperidin-1-oxyl-4-yl octadecanoate (TEMPO-stearate) is used to study the interfacial properties of a variety of phosphatidylcholine vesicles. Since the spin probe exhibits a fast motional electron paramagnetic resonance (EPR) spectrum above the phase transition, the EPR spectrum of the spin probe is analyzed by nonlinear least-squares spectral fitting. EPR spectral line fitting provides high precision spectral parameters, which can be used to construct a detailed picture of the dynamics of the probe and its environment. The hyperfine coupling spacing is used to estimate the effective water concentration in the polar shell of vesicles, while the rotational correlation times give the information on the motion of the spin probe. The effective water concentration of the polar shell of dimyristoyl-phosphatidylglycerol (DMPG) vesicles is greater on average by about 4.0M than the effective water concentration of the polar shell of dimyristoyl-phosphatidylcholine (DMPC) vesicles. The effective water concentration decreases by about 0.5M for an increase of two carbons in the chain, and increases noticeably with hydrocarbon chain unsaturation, which is in good agreement with literature values. The nitroxide moiety rotates preferentially along the N-O bond, that is, parallel to its hydrocarbon chain.     

Impact of coenzyme regeneration on the performance of an enzyme-based optical biosensor: a computational study

A mathematical model of a reagent-less optical sensing scheme composed of an enzymatic reaction coupled to light-controlled photochemical coenzyme regeneration is described. The model is based on previous experimental work describing the regeneration of NADPH from NADP(+) by excited state thionine coupled to the oxidation of isocitrate by isocitrate dehydrogenase. The system is capable of repeated isocitrate measurements without the addition of exogenous coenzyme. The model is simulated using numerical integration to determine the effect of regeneration on the sensor sensitivity, response time and maximum sample throughput rate. Prediction of these effects without a model is difficult due to activation and inhibition of the dehydrogenase by both forms of the coenzyme. The regeneration parameters, including thionine concentration and thionine excitation pattern, are varied to determine optimal sensor conditions to maximize performance. A periodic regeneration approach is found to be superior to a continuous regeneration approach as the former maximizes sensitivity and minimizes response time in most cases. In addition periodic regeneration results in a maximum sample throughput frequency that is achieved at a single optimal thionine level and is independent of the analyte concentration. In contrast the optimal thionine concentration during continuous regeneration varies with the sample analyte concentration. These findings highlight the importance of designing controllable regeneration for dehydrogenase-based optical biosensors.     

荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色效果比较*

比较了荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色的效果。荧光素钠法中样品处理只需20min.左右,具有直接、快速的特点;荧光指示剂对病菌分生孢子萌发及菌丝生长无抑制作用,主要沉集于活菌体的隔膜和细胞质部位,使病菌产生明显的亮绿荧光和清晰的细胞轮廓,亮绿荧光衰退期为7min.;借助荧光显微镜可以观察病菌在小麦叶表的发展过程,区别活菌体和失活菌体。考马斯亮蓝法包括传统的组织学染色步骤,经过改进后的样品处理过程需要40min.左右;染色后使寄主组织呈现淡蓝色,病菌菌体染成深蓝色;该方法可以观察病菌在小麦叶表和被侵染细胞内部发育形成的结构,包括孢子发育形成的初生芽管、附着胞芽管、成熟附着胞以及在寄主细胞内形成的初生吸器原体、成熟的指状体吸器和次生吸器。     

Body composition assessment of young tennis players by multi-frequency impedance measurements

Body composition was estimated in 169 young tennis players using the multi-frequency impedance method to predict their fatfree-mass, fat, extra-cellular water, and total body water. Bioelectrical impedances at 1, 5, 10, 50 and 100 kHz were determined. Ratios of impedance at low to high frequencies (Z1/Z100; Z5/Z100) were also used as indicators of body water distribution. This sample is characterized by a greater body mass — fat mass, above all-than other Italian sport participants. Sexual dimorphism in body composition is also evident in this sample. The parameters of body composition, predicted by impedance measurements were compared with parameters obtained through other methods (skinfolds and BMI) on the same subjects. Results varied considerably, in relation to the various equations or different techniques employed. Besides demonstrating the value of multifrequency impedance measurements in the assessment of body composition, our findings indicate the necessity of further methodological research to validate the method under controlled conditions.     

Theoretical Differential Phase Analysis for Characterization of Aqueous Solution Using Surface Plasmon Resonance

Surface plasmon resonance (SPR)-based differential phase analysis has been presented. Real as well as complex plane analysis of resonance parameters have been undertaken for the optimum selection of metal thicknesses in a bimetallic SPR configuration working under both angular and spectral regime. Theoretically, we can characterize the aqueous solution in terms of this differential phase variation due to the variation of sample parameters such as concentration and temperature. In this respect, two case studies, namely, concentration of hemoglobin in human blood and sensing of temperature of water have been demonstrated and proposed theoretically. By monitoring the change of differential phase, proposed approach leads to a very sensitive measurement of concentration and temperature.