Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH

A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD⁺ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD⁺ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 × 10^?14 mol NADH or NAD⁺ per assay.     

Bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in clinical blood cultures

A bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in 512 clinical aerobic blood cultures was evaluated. At the detection limit of bacterial ATP (10^?10 mol/l) in the blood cultures 94.2% of the true positive blood cultures were detected (sensitivity) and the specificity was 85.8%. If the cut-off limit was increased the sensitivity decreased and the specificity increased and at 2 × 10^?9 mol/l ATP the maximum correctly classified blood cultures was reached. At this cut-off limit the sensitivity was 82.9% and the specificity was 99.6%. In 54.3% of the true positive blood cultures bacterial growth was detected more rapidly with the bioluminescent assay than with macroscopic examination and subculture.     

Functional changes of mouse spermatozoa stored in vitro

In suspensions of epididymal spermatozoa in vitro at +10°C and +37°C, all nuclei-containing and mitochondria-containing structures (normal spermatozoa, spermatozoa with the bent and coiled tails, complexes of head and neck) are with propidium iodide and rhodamine 123, respectively. Intracellular ATP concentration determined by a bioluminescent method in mitochondria-containing elements of suspension decreases (significantly faster at 37°C than at 10°C) up to a certain unchangeable level (2.5 × 10^?8 M/l at 37°C and to 1.6 × 10^?8 M/l at 10°C per 10⁶ of mitochondria-containing elements). Mechanisms of spermatozoa destruction are discussed.     

A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter

The recombinant Ca²⁺-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10^?15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample.     

Chemiluminescent assay of co-factors

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperox-idase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 × 10^?14 to 5 × 10^?12mol/assay. The detection limit of NADH was 30fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.     

Bioluminescent assay of D-3-hydroxybutyrate in serum

An enzymatic assay of D -3-hydroxybutyrate in which the hydroxybutyrate dehydrogenase reaction is coupled to the bacterial oxidoreductase—uciferase system is described. The bioluminescent assay is based on either, end-point, or on initial velocity measurements. This simple and rapid assay requires a single serum sample of 10 μl. Its linear range covers two orders of magnitude from 10^?6 mol/I upwards. This assay is suitable for the routine determination of D -3-hydroxybutyrate in human blood with good accuracy.     

Bioluminescent eddies of the World Ocean

Mesoscale eddies of the ocean (with a characteristic diameter of about 100 km and a life time-span of about several weeks) are habitats of plankton organisms, many of which are bioluminescent. The spatial heterogeneity of bioluminescence of the upper mixed layer associated with the impact of mesoscale eddies is poorly studied. The 45-year historical data set was retrieved, in order to select the bathy-photometric surveys carried out in the form of station grids and transects across eddies. Data from 71 expeditions deployed in 1966–2022 to the Atlantic Ocean, Indian Ocean and Mediterranean Sea basin were analyzed, in order for the spatial heterogeneity of bioluminescent fields to be elucidated across eddy fields. The stimulated bioluminescence intensity was characterized by the bioluminescent potential, which represented the maximal amount of radiant energy emitted in a given volume of water by bioluminescent organisms. The normalized bioluminescent potential over oceanographic station grids exhibited correlation with the eddy kinetic energy and zooplankton biomass (r = 0.8, at P = 0.001 and r = 0.7, at P = 0.05, respectively), in a broad range of energy and bioluminescence units (0.02–0.2 m² s⁻²; 0.4–92.0 × 10⁻⁸ W cm⁻² L⁻¹, respectively). Overall, estimates of bioluminescent potential variability on the mesoscale contribute to the assessment of the multiple-scale variation of the bioluminescent field of the World Ocean.     

Action spectrum for a low intensity, rapid photoinhibition of mechanically stimulable bioluminescence in the marine dinoflagellates Gonyaulax catenella, G. acatenella, and G. tamarensis

The mechanically stimulable bioluminescence of members of the Gonyaulax catenella group can be maximally photoinhibited by exposure to as few as 10¹³ quanta/cm², a factor 10⁴ times smaller than that required for comparable photoinhibition in Gonyaulax polyedra and all other photosynthetic bioluminescent dinoflagellates investigated. Following an irradiation pulse there is an initial time lag of one minute, followed by a rapid decrease in mechanical stimulability to approximately 1% of the dark unirradiated control with a firstorder rate constant as high as 0.01 sec^?1. Action spectra for all three species imply a pigment with a single absorption band having a maximum at 562 nm and a half band width of 105 nm within the spectral range 325 nm to 775 nm. Photoinhibition appears to decrease either the sensitivity of the shear receptor mechanism or the efficiency of signal transmission in the dinoflagellates, since chemically stimulable bioluminescence is unaffected by these exposures.     

Detection of Hispidin by a Luminescent System from Basidiomycete <Emphasis Type="Italic">Armillaria borealis</Emphasis>

In in vitro experiments, the possibility of using a luminescent system extracted from the luminous fungus Armillaria borealis has been shown to detect and determine the concentration of hispidin. A linear dependence of the luminescent response on the content of hispidin in solutions in the concentration range of 5.4 × 10^–5–1.4 × 10^–2 µM was detected. The stability of the enzyme system and the high sensitivity of the bioluminescent reaction allows carrying out multiple measurements with the analyte detection limit of 1.3 × 10^–11 g. The obtained results show the prospects of creating a rapid bioluminescent method for the analysis of medical substances or extracts from various biological objects for the presence of hispidin.     

A rapid sensitive method for the measurement of guanine ribonucleotides in bacterial and environmental extracts.

A method was devised for the quantitative determination of guanine ribonucleotides (GTP, GDP, and GMP) in extracts of biological materlals. The technique is based upon selective enzymatic hydrolysis of UTP and ATP contained within the cell extracts, followed by a quantitative determination of GTP. GTP is measured using a nucleoside diphosphate kinase-firefly luciferase coupled bioluminescent reaction, during which the GTP is enzymatically coupled to ATP production, resulting in ATP-dependent light emission. The methods are simple and reproducible and extremely sensitive (≤ 10^?9m GTP), and require no preparatory chromatographic separation procedures. Methods are also presented for the enzymatic conversions of GDP and GMP to GTP in addition to the determination of GTP.     

SPECIES OF OCEANIC DINOFLAGELLATES IN THE GENERA DISSODINIUM AND PYROCYSTIS: INTERCLONAL AND INTERSPECIFIC COMPARISONS OF THE COLOR AND PHOTON YIELD OF BIOLUMINESCENCE1

We have examined aspects of the bioluminescence of 5 clones of Dissodinium, 1 clone of Pyrocystis acuta, 4 clones of Pyrocystis fusiformis, and 5 clones of Pyrocystis noctiluca. All clones produced the same color bioluminescence with an intensity peak near 474 nm. The in vivo emission spectra of these clones agreed with those previously determined, for 4 other species of marine dinoflagellates. The amount of light emitted by the dinoflagellates in scotophase when mechanically stimulated to exhaustion was determined for most of the clones. The largest species, P. noctiluca and P. fusiformis, emitted 37–89 × 10⁹ photons cell^?1 and 23–62 × 10⁹ photons cell^?1, respectively, about a thousand, times as much light as Gonyaulax species. Pyrocystis acuta emitted 3–6 × 10⁹ photons cell^?1. Three of the 5 clones of Dissodinium were bioluminescent. The range for 3 clones was 5–13 × 10⁹ photons cell^?1. All 5 clones of Dissodinium are morphologically distinct. Both the clones of Dissodinium and Pyrocystis produced much higher numbers of photons per cell nitrogen (ca. 7–50 times) than Gonyaulax polyedra or Pyrodinium bahamense. The data suggested that enzyme turnover occurred in the reactions producing light during mechanical stimulation of Dissodinium and Pyrocystis species.     

A flow calorimeter for assay of hormone- and metabolite-induced changes in steady-state heat production by tissue

A compact, heat conduction, flow calorimeter for use in monitoring tissue response to metabolic regulators has been designed and constructed. The instrument operates as a perfusion apparatus. Heat production by tissue can be measured continuously, and nutrient and oxygen concentrations can be kept at optimum, constant levels. Introduction of substances of interest is made without production of attendant thermal mixing artifacts. The resolution time of events is less than 10 s. The thermal stability of the instrument is maintained by dynamic methods. The sensitivity of the instrument, 7.2 μW/μV, allows observation of changes in heat production as small as 3 × 10^?4 to 5 × 10^?4 cal/min, the equivalent of 3–5% of the heat production of a representative 1-g quantity of fresh tissue. The calorimeter was used to monitor changes in the steady-state heat production of 250-mg samples of corn coleoptile tissue in response to the plant growth substance, indoleacetic acid.     

EFFECTS OF LIGHT INTENSITY ON DIVISION RATE,STIMULABLE BIOLUMINESCENCE AND CELL SIZE OF THE OCEANIC DINOFLAGELLATES DISSODINIUM LUNULA,PYROCYSTIS FUSIFORMIS AND P. NOCTILUCA12

Preadapted cultures were grown in a 12:12 LD cycle at a series of light intensities under cool-white, fluorescent lamps. Pyrocystis fusiformis Murray maintained high division rates at low light intensities at the expense of cell size. In contrast, Dissodinium lunula (Schuett) Taylor had relatively lower division rates at low light intensities with little concomitant decrease in size. The response of P. noctiluca Murray was intermediate between these two species. For all three, cell numbers did not increase above an intensity of 5–10 μEin·m^?2·sec^?1 and division rate was saturated at ca. 30, 60, and 60μEin·m^?2·sec^?1 for P. fusiformis, P. noctiluca, and D. lunula, respectively. The capacity for stimulable bioluminescence was saturated at light intensities of 0.15 μEin·m^?2·day in short-term (2-day) experiments. In cultures of P. fusiformis and P. noctiluca, maintained for at least one month at lower intensities than needed to saturate division rate, a decrease in the capacity for stimulable bioluminescence was accompanied by a reduction in cell size. Our results suggest that cell size and bioluminescent capacity may prove to be a potentially useful indication of the history of exposure of natural populations of Pyrocystis spp. to ambient intensities.     

Simultaneous determination of ΔG, ΔH and ΔS by an automatic microcalorimetric titration technique. Application to protein ligand binding

A methodological study has been made with a syringe titration unit attached to an LKB batch microcalorimeter. The presicion and accuracy of the instrument assembly have been evaluated by neutralization reactions and by dilution of sucrose solutions. As an example, heat quantities on the order of 10 mJ accompanying the addition of 10 μl titrant solution could be determined with an accuracy of better than 1%. A stepwise titration procedure was used to characterize the binding of indole-3-propionic acid to α-chymotrypsin. The following thermodynamic data were obtained (25°C, acetate buffer, pH 5.80): ΔG⁰ = ?18.46±0.17 kJ·mol^?1, ΔH⁰ = ?15.26±0.20 kJ·mol^?1, ΔS⁰ = 10.85±1.21 JK^?·mol^?1.     

Design of a phosphinate‐based bioluminescent probe for superoxide radical anion imaging in living cells

Superoxide radical anion (O₂˙^?) as an important member of reactive oxygen species (ROS) plays a vital role both in physiology and pathology. Herein we designed and synthesized a novel phosphinate‐based bioluminescence probe for O₂˙^? detection in living cells, which exhibited good sensitivity for capturing O₂˙^? at the nanomole level and high selectivity against other ROS. The probe was further found to be of low toxicity for living cells and was then successfully employed for sensing endogenous O₂˙^? by using phorbol‐12‐myristate‐13‐acetate (PMA) as a traditional O₂˙^? stimulator in Huh7 cells. Moreover, the increasing production and use of nanoparticles, has given rise to many concerns and debates among the public and scientific authorities regarding their safety and final fate in biological systems. Herein it was found that mondisperse polystyrene particles could stimulate O₂˙^? generation in Huh7 cells. Overall, the probe was demonstrated to have a great potential as a novel bioluminescent sensor for detecting O₂˙^? in living cells. To our knowledge, this is the first small‐molecule phosphinate‐based bioluminescence probe that will open up great opportunities for unlocking the mystery of O₂˙^? in human health and disease.     

Continuous in situ monitoring of sediment deposition in shallow benthic environments

Sedimentation is considered the most widespread contemporary, human-induced perturbation on reefs, and yet if the problems associated with its estimation using sediment traps are recognized, there have been few reliable measurements made over time frames relevant to the local organisms. This study describes the design, calibration and testing of an in situ optical backscatter sediment deposition sensor capable of measuring sedimentation over intervals of a few hours. The instrument has been reconfigured from an earlier version to include 15 measurement points instead of one, and to have a more rugose measuring surface with a microtopography similar to a coral. Laboratory tests of the instrument with different sediment types, colours, particle sizes and under different flow regimes gave similar accumulation estimates to SedPods, but lower estimates than sediment traps. At higher flow rates (9–17 cm s^?1), the deposition sensor and SedPods gave estimates >10× lower than trap accumulation rates. The instrument was deployed for 39 d in a highly turbid inshore area in the Great Barrier Reef. Sediment deposition varied by several orders of magnitude, occurring in either a relatively uniform (constant) pattern or a pulsed pattern characterized by short-term (4–6 h) periods of ‘enhanced’ deposition, occurring daily or twice daily and modulated by the tidal phase. For the whole deployment, which included several very high wind events and suspended sediment concentrations (SSCs) >100 mg L^?1, deposition rates averaged 19 ± 16 mg cm^?2 d^?1. For the first half of the deployment, where SSCs varied from <1 to 28 mg L^?1 which is more typical for the study area, the deposition rate averaged only 8 ± 5 mg cm^?2 d^?1. The capacity to measure sedimentation rates over a few hours is discussed in terms of examining the risk from sediment deposition associated with catchment run-off, natural wind/wave events and dredging activities.     

Impact of cyanogen iodide in killing of Escherichia coli by the lactoperoxidase-hydrogen peroxide-(pseudo)halide system

In the presence of hydrogen peroxide, the heme protein lactoperoxidase is able to oxidize thiocyanate and iodide to hypothiocyanite, reactive iodine species, and the inter(pseudo)halogen cyanogen iodide. The killing efficiency of these oxidants and of the lactoperoxidase-H₂O₂-SCN^?/I^? system was investigated on the bioluminescent Escherichia coli K12 strain that allows time-resolved determination of cell viability. Among the tested oxidants, cyanogen iodide was most efficient in killing E. coli, followed by reactive iodine species and hypothiocyanite. Thereby, the killing activity of the LPO-H₂O₂-SCN^?/I^? system was greatly enhanced in comparison to the sole application of iodide when I^? was applied in two- to twenty-fold excess over SCN^?. Further evidence for the contribution of cyanogen iodide in killing of E. coli was obtained by applying methionine. This amino acid disturbed the killing of E. coli mediated by reactive iodine species (partial inhibition) and cyanogen iodide (total inhibition), but not by hypothiocyanite. Changes in luminescence of E. coli cells correlate with measurements of colony forming units after incubation of cells with the LPO-H₂O₂-SCN^?/I^? system or with cyanogen iodide. Taken together, these results are important for the future optimization of the use of lactoperoxidase in biotechnological applications.     

First results obtained with a sweeping pulsed radar reflectometer in the Globus-M tokamak

A weeping pulsed radar reflectometer designed for measuring the spatial electron density distribution in the Globus-M spherical tokamak with a minor plasma radius of a=24 cm, a major radius of R=36 cm, a toroidal field of B _T=0.5 T, a plasma current of I _p=200 kA, and an average density of n=(3–10)×10¹³ cm^?3 is described. The reflectometer operation is based on the reflection of microwaves with a carrier frequency f from a plasma layer with the critical density n=(0.0111f)², where n is the electron density in units of 10¹⁴ cm^?3 and f is the microwave frequency in GHz. By simultaneously probing the plasma at different frequencies, it is possible to recover the electron density profile. Microwave pulses with different frequencies are obtained by frequency sweeping. To increase the range of measured densities, channels with fixed frequencies are also used; as a result, the instrument has eleven frequency channels: a 19.5-GHz channel, eight channels in the 26-to 40-GHz frequency range, a 51.5-GHz channel, and a 60-GHz channel, which corresponds to eleven points in the density profile: 0.47×10¹³ cm^?3, eight points in the (0.8–1.95)×10¹³-cm^?3 range, 3.27×10¹³ cm^?3, and 4.5×10¹³ cm^?3. The reflectometer allows detailed measurements of the density profile with a time resolution of several tens of microseconds, which can be useful, in particular, in studying the processes related to the formation of an internal transport barrier in plasma. The first results obtained using this reflectometer in the Globus-M tokamak under various operating conditions are discussed.     

A Bioluminescent Whole-Cell Reporter for Detection of 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol in Soil

A bioreporter was made containing a tfdRP_DII-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (R² = 0.9825) in the range of 2.0 μM to 1.1 × 10² μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 10² μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.