Determination of oxygen gradients in engineered tissue using a fluorescent sensor

Nutrient and oxygen supply of cells are crucial to tissue engineering in general. If a sufficient supply cannot be maintained, the development of the tissue will slow down or even fail completely. Previous studies on oxygen supply have focused on measurement of oxygen partial pressures (pO(2)) in culture media or described the use of invasive techniques with spatially limited resolution. The experimental setup described here allows for continuous, noninvasive, high-resolution pO(2) measurements over the cross-section of cultivated tissues. Applying a recently developed technique for time-resolved pO(2) sensing using optical sensor foils, containing luminescent O(2)-sensitive indicator dyes, we were able to monitor and analyze gradients in the oxygen supply in a tissue over a 3-week culture period. Cylindrical tissue samples were immobilized on top of the sensors. By measuring the luminescence decay time, two-dimensional pO(2) distributions across the tissue section in contact with the foil surface were determined. We applied this technique to cartilage explants and to tissue-engineered cartilage. For both tissue types, changes were detected in monotonously decreasing gradients of pO(2) from the surface with high pO(2) to minimum pO(2) values in the center of the samples. Nearly anoxic conditions were observed in tissue constructs ( approximately 0 Torr) but not in excised cartilage discs ( approximately 20 Torr) after 1 day. Furthermore, the oxygen supply seemed to strongly depend on cell density and cell function. Additionally, histological analysis revealed a maximum depth of approximately 1.3 mm of regular cartilage development in constructs grown under the applied culture conditions. Correlating analytical and histological analysis with the oxygen distributions, we found that pO(2) values below 11 Torr might impair proper tissue development in the center. The results illustrate that the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures.     

Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen

Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.     

A comparative study of fluorescent singlet oxygen probes in plant leaves

Four fluorescent singlet oxygen sensors: DanePy, its oxalate salt, Singlet Oxygen Sensor Green and MVP, were infiltrated into tobacco leaves and tested for toxicity, subcellular localization, light sensitivity and capacity to trap the singlet oxygen produced in photoinhibition. For reference, a broad sensitivity free radical probe, TEMPO-9-AC, was also included. Photochemical yield was approximately 15% and 10% inhibited by Singlet Oxygen Sensor Green and MVP, respectively, but was not significantly affected by the other probes. Under photoinhibitory conditions, brought about by irradiating lincomycin-treated leaves with strong photosynthetically active radiation, DanePy and Singlet Oxygen Sensor Green were responsive. Singlet Oxygen Sensor Green was also reactive to low, non-photoinhibitory light exposure of the leaf, which was not characteristic to the other probes. MVP did not respond to singlet oxygen which can partly be explained by a possible attenuation of its blue emission in the leaf, as shown by the example TEMPO-9-AC. DanePy-oxalate did not respond to photosynthetic singlet oxygen due to lack of its penetration into photosynthetic tissue and hence could be useful in detecting any singlet oxygen which escapes from a chloroplast initiation site. DanePy was localized in the chloroplasts, while Singlet Oxygen Sensor Green was mainly found in the epidermal cells preferentially associated with the nucleus.      

Effect of proline on the production of singlet oxygen

Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (1O2) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of 1O2 with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.     

Singlet oxygen production from the reactions of ozone with biological molecules

The reaction of ozone with a number of biological molecules was found to produce singlet oxygen in high yield. At pH 7.0, the reaction of ozone with an equimolar amount of biological molecule produced the following singlet oxygen yields (mole of singlet oxygen/mole of ozone): cysteine, 0.49 +/- 0.02; methionine, 1.13 +/- 0.11; reduced glutathione, 0.33 +/- 0.02; albumin, 1.00 +/- 0.05; uric acid, 0.64 +/- 0.09; ascorbic acid, 0.96 +/- 0.007; NADPH, 1.07 +/- 0.07; NADH, 0.95 +/- 0.01. Thus, singlet oxygen may be an important intermediate in the biochemical damage caused by ozone.     

Imaging dynamic cell signaling in vivo with new classes of fluorescent reporters

Dynamical features of cell signaling are the essence of living organisms. To understand animal development, it is fundamental to investigate signaling dynamics in vivo. Robust reporters are required to visualize spatial and temporal dynamics of enzyme activities and protein–protein interactions involved in signaling pathways. In this review, we summarize recent development in the design of new classes of fluorescent reporters for imaging dynamic activities of proteases, kinases, and protein–protein interactions. These reporters operate on new physical and/or chemical principles; achieve large dynamic range, high brightness, and fast kinetics; and reveal spatiotemporal dynamics of signaling that is correlated with developmental events such as embryonic morphogenesis in live animals including Drosophila and zebrafish. Therefore, many of these reporters are great tools for biological discovery and mechanistic understanding of animal development and disease progression.     

Inhibition of Photosystem II by the singlet oxygen sensor compounds TEMP and TEMPD

2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethyl-4-piperidinone (TEMPD) have earlier been used to quantify singlet oxygen produced by plant material. Both compounds were found to cause severe side effects on Photosystem II. Addition of TEMP or TEMPD to thylakoids immediately stabilized the reduced state of the Q(A) electron acceptor and destabilized the reduced state of the Q(B) acceptor, causing decrease in the driving force of forward electron transfer. Oxygen evolution, thermoluminescence and fluorescence measurements indicated that the number of functional PSII units decreased during incubation of thylakoids with TEMP or TEMPD. Singlet oxygen determinations in photosynthetic systems with piperidine derivatives should be interpreted with care.     

Singlet oxygen production from the peroxidase-catalyzed oxidation of indole-3-acetic acid

The aerobic oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase produces 1268 nm emission characteristic of singlet oxygen. Lactoperoxidase also oxidizes indole-3-acetic acid to produce singlet oxygen, but in contrast to horseradish peroxidase, this enzyme system requires hydrogen peroxide. In both of these systems, the intensity of the 1268 nm emission is small due to quenching of the singlet oxygen by indole-3-acetic acid and by reaction products derived from indole-3-acetic acid. The biomolecular reaction of peroxyl radicals via a Russell mechanism is a plausible mechanism for the singlet oxygen generation in these systems. Under typical conditions of p2H 4.0, 1 microM horseradish peroxidase, 1 mM indole-3-acetic acid, and 240 microM oxygen, the singlet oxygen yield was 15 +/- 1 microM or 13% of the amount predicted by the Russell mechanism.     

Green fluorescent protein as a new expression marker in mycobacteria

This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM-11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM-11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co-factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free-living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.     

Singlet oxygen production from the peroxidase catalyzed formation of styrene glutathione adducts

Recently, Stock et al. (J. Biol. Chem. 261, 15915-15922 [1986]) described a model enzyme system composed of horseradish peroxidase, hydrogen peroxide, phenol, glutathione and styrene. This system forms glutathione-styrene conjugates. Glutathione radicals and carbon-centered radicals are intermediates in this process. In the present study, this model enzyme system was also shown to generate singlet oxygen, probably via a Russell mechanism. No singlet oxygen was generated in the absence of styrene. Thus, contrary to prior suggestions, the reaction of glutathione radical with oxygen to produce a thiyl peroxyl radical is not a significant source of singlet oxygen.     

Measurement of photosynthetic oxygen evolution with a new type of oxygen sensor

We measured the light response curve of photosynthetic oxygen evolution by illuminating a leaf disc in an air-tight windowed chamber. Oxygen production was measured by monitoring the quenching of luminescence of an organometallic ruthenium compound. A photodiode based chlorophyll a fluorometer was used to measure the luminescence intensity. Oxygen evolution measurements with a traditional oxygen electrode gave the same numerical values at different light intensities when the same leaf disk was tested. The quality of the measurement signal of the new method was found to be similar to that obtained with the oxygen electrode method. The new luminescence based system is more stable against electrical disturbances than an oxygen electrode, its response to oxygen pressure changes is very rapid, and the new method allows the same basic equipment to be used for chlorophyll fluorescence and oxygen measurements.     

Cytochrome c enhancement of singlet molecular oxygen production by the NADPH-dependent adrenodoxin reductase-adrenodoxin system: the role of singlet oxygen in damaging adrenal mitochondrial membranes

In the presence of NADPH, cytochrome c stimulates approximately a 200-fold increase in the production of singlet oxygen by the bovine adrenodoxin reductase-adrenodoxin system. The formation of singlet oxygen, which was monitored by the attending chemiluminescence, was markedly inhibited by the addition of superoxide dismutase or 1,4-diazabicyclo[2.2.2]octane. The adrenal system, in the presence of cytochrome c, peroxidized adrenal mitochondrial lipids, as indicated by the formation of malondialdehyde. This oxidation is also inhibited by the addition of dismutase and 1,4-diazabicyclo[2.2.2]octane.     

A study of oxygen transfer in shake flasks using a non-invasive oxygen sensor

We describe a study of oxygen transfer in shake flasks using a non-invasive optical sensor. This study investigates the effect of different plugs, presence of baffles, and the type of media on the dissolved oxygen profiles during Escherichia coli fermentation. We measured the volumetric mass transfer coefficient (k(L)a) under various conditions and also the resistances of the various plugs. Finally, we compared shake flask k(L)a with that from a stirred tank fermentor. By matching k(L)a's we were able to obtain similar growth and recombinant protein product formation kinetics in both a fermentor and a shake flask. These results provide a quantitative comparison of fermentations in a shake flask vs. a bench-scale fermentor and should be valuable in guiding scale-up efforts.     

Production of singlet oxygen in a non-self-sustained discharge

The production of O₂(a¹Δ_g) singlet oxygen in non-self-sustained discharges in pure oxygen and mixtures of oxygen with noble gases (Ar or He) was studied experimentally. It is shown that the energy efficiency of O₂(a¹Δ_g production can be optimized with respect to the reduced electric field E/N. It is shown that the optimal E/N values correspond to electron temperatures of 1.2–1.4 eV. At these E/N values, a decrease in the oxygen percentage in the mixture leads to an increase in the excitation rate of singlet oxygen because of the increase in the specific energy deposition per O₂ molecule. The onset of discharge instabilities not only greatly reduces the energy efficiency of singlet oxygen production but also makes it impossible to achieve high energy deposition in a non-self-sustained discharge. A model of a non-self-sustained discharge in pure oxygen is developed. It is shown that good agreement between the experimental and computed results for a discharge in oxygen over a wide range of reduced electric fields can be achieved only by taking into account the ion component of the discharge current. The cross section for the electron-impact excitation of O₂(a¹Δ_g and the kinetic scheme of the discharge processes with the participation of singlet oxygen are verified by comparing the experimental and computed data on the energy efficiency of the production of O₂(a¹Δ_g and the dynamics of its concentration. It is shown that, in the dynamics of O₂(a¹Δ_g molecules in the discharge afterglow, an important role is played by their deexcitation in a three-body reaction with the participation of O(³P) atoms. At high energy depositions in a non-self-sustained discharge, this reaction can reduce the maximal attainable concentration of singlet oxygen. The effect of a hydrogen additive to an Ar: O₂ mixture is analyzed based on the results obtained using the model developed. It is shown that, for actual electron beam current densities, a significant energy deposition in a non-self-sustained discharge in the mixtures under study can be achieved due to the high rate of electron detachment from negative ions. In this case, however, significant heating of the mixture can lead to a rapid quenching of O₂(a¹Δ_g molecules by atomic hydrogen.     

Oxygen monitoring in supercritical carbon dioxide using a fibre optic sensor

Investigations of enzymatic reactions in supercritical CO(2) are often hindered by the high pressure involved in these processes, making reaction monitoring extremely difficult. This paper describes the implementation of a fiber optic based oxygen sensor into a high pressure reactor for supercritical carbon dioxide. The sensor is pressure resistant, working in supercritical carbon dioxide and reusable after depressurisation. The sensor signal is found to be affected by pressure changes, but stable at constant pressure. Oxygen concentration in supercritical CO(2) is monitored using the disproportionation of hydrogen peroxide as a simple oxygen producing reaction.     

Reactivities of diphenylfuran (a singlet oxygen trap) with singlet oxygen and hydroxyl radical in aqueous systems.

It has been studied whether 2,5-diphenylfuran is a specific singlet oxygen trap in aqueous systems. With certain ¹O₂ generating systems (Rose Bengal photooxygenation and NaOClH₂O₂ systems) and·OH generating systems (Fenton's reagent and acetaldehyde-xanthine oxidase system), diphenylfuran was chiefly converted in all cases to cis-dibenzoyl-ethylene, but not to trans-dibenzoylethylene. Low but detectable conversion of diphenylfuran to a hydroperoxide, probably a distinct ¹O₂-derived reaction in aqueous media, was found only in the Rose Bengal photooxygenation system.     

Effect of ascorbic acid on the production of singlet oxygen by purified human myeloperoxidase

We have previously studied purified human myeloperoxidase-hydrogen peroxide-halide ion systems as models of possible singlet oxygen production by granulocytes. While myeloperoxidase could efficiently produce singlet oxygen, the yield of singlet oxygen at a physiological pH with Cl- was very small due to enzyme inactivation. In that Bolscher et al. [(1984) Biochim. Biophys. Acta 784, 189-191] observed that micromolar concentrations of ascorbic acid prevented inactivation of myeloperoxidase and increased the production of hypochlorous acid, we examined whether ascorbic acid would augment singlet oxygen production by the myeloperoxidase-hydrogen peroxide-halide ion systems. Ascorbic acid, however, fails to increase the singlet oxygen yield, suggesting that it does not augment singlet oxygen production in the intact granulocyte by a myeloperoxidase-dependent mechanism.     

绿荧光蛋白及其在转基因动物研究中的应用

绿荧光蛋白（ｇｒｅｅｎｆｌｕｏｒｅｓｃｅｎｔｐｒｏｔｅｉｎ,ＧＦＰ）是源于水母（Ｊｅｌｙｆｉｓｈ）、海笔（ＳｅａＰｅｎ,ＳｅａＰａｎｓｙ）等海洋无脊椎动物的一种蛋白质,这种蛋白质在体外经适当波长的光激发便可发出绿光,所发出的绿光用普通荧光显微镜或荧光激活细胞分拣器（ＦＡＣＳ）均可检测到。ＧＦＰ作为动、植物以及微生物基因工程研究上的一种选择标记具有检测灵敏度高,操作简便,对机体毒副作用小且不需要添加任何底物或辅助因子等优点,更重要的是检测ＧＦＰ无损于细胞或胚胎的完整性及活力。本文概括介绍ＧＦＰ的生化、发光光谱及遗传学特征及其在转基因动物研究上的应用。