Stable-light-emitting Escherichia coli as a biosensor

We have studied possibilities for constructing Escherichia coli strains capable of producing stable light. Light production in E. coli is achieved by cloning the genes encoding bacterial luciferase from Vibrio harveyi. To gain the advantage of sensitive detection of light we transferred the genes under the control of a strong, regulatable promoter system. Stabilization of light produced by E. coli clones was accomplished by finding the optimal plasmid construction and growth conditions as well as suitable measuring buffers. The adjustment of the luciferase synthesis for bioluminescence measurements to a high but not harmful level gives healthy cells and stable luciferase. Cultivation at 30 °C in an uninduced state was found to be the most important factor in getting stable-light production. The overall cell metabolism being unstressed gives us the possibility of monitoring cell physiology and factors affecting it via bioluminescence reactions in vivo. To make the results easy to interpret the light emission has to be stable during a measurement period of one to several hours. In the case of the original light-producing bacteria, Vibrio and Photobacterium strains it has not thus far been possible to find conditions where light emission would be stable for several hours. Based on our findings an automated biosensor system can be developed to monitor the effects of biologically active compounds against stable-light-producing bacteria.     

Discrimination between different types of low-level luminescence in mammalian cells: The biophysical radiation

Cellular low-level luminescence was measured after various disintegrative processes in brain cell preparations. In addition to known origins of low-level luminescence, e.g. oxygen radical reactions or enzymatic and non-enzymatic redox systems, a further source of photon emission is reported which is independent of external oxygen, oxygen radicals and enzyme activities. Vital cells from rat brain homogenates or pig oligodendrocytes could be kept for hours at 37 °C without any photon emission. Only after disintegrative processes a cellular photon emission could be induced. The maximal intensity of about 400 impulses/s/mg protein and a total radiation of about 6 × 10⁶ I/mg depended on the type of cells. The signal could be retained completely at 4 °C or in frozen samples. Heating (10 min, 90 °C) did not suppress the photon emission. Luminol and lucigenin did not amplify the signal as is usually observed in oxygen radical-producing cells. Non-specific radical scavengers as well as detergents suppressed the cellular photon emission completely. It is suggested that this cellular luminescence represents a biophysical radiation which originates from the interruption of an intermolecular radiationless energy transfer.     

External electric field effects on photosynthetic membrane vesicles. Kinetic characterization of two electrophotoluminescence phases in hypotonically swollen chloroplasts

Strong externally applied electrical field pulses are known to stimulate delayed luminescence from preilluminated blebs (hypotonically swollen vesicles originating from thylakoid membranes of broken chloroplasts) by up to 3 orders of magnitude. This phenomenon is known as electrophotoluminescence. Previous analysis showed the kinetics of the electrophotoluminescence to be biphasic, displaying a rapid (R) phase which decays towards a slower one (S) (Ellenson, J.L. and Sauer, K. (1976) Photochem. Photobiol. 23, 113–123). We demonstrate that these two components represent different processes. At low pH, a good kinetic separation is obtained between the two phases, which become distinct, with the S phase manifesting also an initial rise period. Under these conditions, it is possible to estimate separately the approximate rise times of the two phases. It is shown that the R and S components have a different dependence on the pH and on the time between the actinic flash and onset of the field. The field dependence is also different, with the S phase requiring a lower threshold field than R. From these observations, it is concluded that the R and S luminescence components are formed by different precursors. The difference in behaviour of the two phases during formation of the bleb indicates that the precursors of the R and S phases belong to different parts of the bleb. We suggest that R precursors are located in the wall of the swollen thylakoid and S precursors in the membrane formations which are attached to this wall.     

Magnesium-dependent induction of phagocytosis-associated chemiluminescence of adherent human polymorphonuclear leukocytes by non-opsonized zymosan

A severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) to non-opsonized zymosan was observed under a deficiency of extracellular Mg²⁺. The phagocytosis-association native (luminol-independent) luminescence (NL), as well as luminol-dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongely inhibited. Apart from a general decrease of total light production, a Mg²⁺-concentration-dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane-bound NADPH-oxidase of PMNL is suggested. The presence of extracellular Ca²⁺ did not compensate for the Mg²⁺ deficit. In the presence of Mg²⁺ only a slight Ca²⁺-dependent reduction of NL was obtained, but Ca²⁺ seemed to selectively promote LDL. This may indicate a positive influence of Ca²⁺ on the myeloperoxidase release from the cells. Experiments with the metalions-chelating agents EDTA and EGTA, which complex Mg²⁺ to differing extents, confirmed the important role of Mg²⁺ in PMNL-activation by non-opsonized zymosan.     

Time‐resolved spectral measurements of delayed luminescence from a single soybean seed: effects of thermal damage and correlation with germination performance

Delayed luminescence from a single dry soybean seed was investigated in both spectral and time domains, under different excitation wavelengths. Emission spectra were collected, under 337 nm laser excitation, from native and artificially deteriorated seeds and the time‐dependence of different spectral components was analyzed in detail. The single seed viability was evaluated through observation of germination properties after imbibition and compared with different parameters related to the luminescence kinetics. The significant correlation found between single seed delayed luminescence parameters and germination capability strongly validates the connection of this phenomenon with the functional state of the system and suggests the development of a non‐invasive technique for seed quality determination. Copyright © 2009 John Wiley & Sons, Ltd.     

Cuscuta reflexa invasion induces Ca2+ release in its host

Cuscuta reflexa induces a variety of reaction in its hosts. Some of these are visual reactions, and it is clear that these morphological changes are preceded by events at the molecular level, where signal transduction is one of the early processes. Calcium (Ca²⁺) release is the major second messenger during signal transduction, and we therefore studied Ca²⁺ spiking in tomato during infection with C. reflexa. Bioluminescence in aequorin‐expressing tomato was monitored for 48 h after the onset of Cuscuta infestation. Signals at the attachment sites were observed from 30 to 48 h. Treatment of aequorin‐expressing tomato leaf disks with Cuscuta plant extracts suggested that the substance that induced Ca²⁺ release from the host was closely linked to parasite haustoria.     

Luminescence studies on RbI:Tb3+ crystals.

Optical absorption, photoluminescence, thermoluminescence (TL) and photostimulated luminescence (PSL) studies on RbI:Tb(3+) crystals irradiated with gamma-rays is reported. Photoluminescence of these crystals exhibits characteristic Tb(3+) emissions, due to transitions from the (5)D(3) and (5)D(4) levels to various levels of the (7)F septet. On F-bleaching the gamma-irradiated crystals, Z(3) centres are observed. The TL glow curve indicates a two-step thermal annihilation process for the radiatively created defects. The presence of the characteristic emissions due to terbium ions in the photostimulation at the F-band, and TL emissions under both glow peaks, confirm the participation of Tb ions in the defect production and recombination processes. Trap parameters for the TL process are calculated and presented. The low temperature glow peak is attributable to Z(3) centres.     

Luminescence properties of neodymium,samarium, and europium niobate and tantalate thin films

Thin films of lanthanide orthoniobate LnNbO₄ (LnNO) and orthotantalate LnTaO₄ (LnTO), (Ln = Nd, Sm, Eu) were fabricated using the sol–gel method with subsequent spin-coating on the PbZrO₃/Al₂O₃ substrate and annealing at 1000°C. X-ray diffraction patterns showed monoclinic M-LnNbO₄ or M´-LnTaO₄, which coexists with the orthorhombic or tetragonal phase. X-ray photoelectron spectroscopy demonstrated the presence of Nd³⁺, Sm³⁺/Sm²⁺ and Eu³⁺/Eu²⁺ ions. The luminescence properties of polymorphic films were investigated. Excitation spectra of PbZrO₃ interlayer represented broad bands at 410 and 550 nm that were assigned to charge transfer bands (CTB). In all films, the CTB broad band at ~275 nm related to charge transfer transition of Ln³⁺→O²⁻ and NbO₄³⁻ or TaO₄³⁻ groups. In excitation spectra, ⁴I_9/2→⁴G_5/2 (Nd³⁺), ⁶H_5/2→⁶P_3/2 (Sm³⁺) and ⁷F₀→⁵L₆ (Eu³⁺) transitions (at 585, 402 and 395 nm), respectively were found to be more intense than any other Ln³⁺ transition. The emission spectra showed narrow and intense bands at 1065, 600, and 614 nm that were ascribed to Nd³⁺, Sm³⁺, and Eu³⁺ 4f–f intraconfigurational transitions ⁴F_3/2→⁴I_11/2, ⁴G_5/2→⁶H_7/2, and ⁵D₀→⁷F₂, respectively. The excellent luminescence properties of films make them new potential groups for visible and/or near-infrared applications such as sensors and imaging equipment.