Quenching of Chlorophyll a Fluorescence in Response to Na+-Dependent HCO3- Transport-Mediated Accumulation of Inorganic Carbon in the Cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the yield of chlorophyll a fluorescence decreased in response to the transport-mediated accumulation of intracellular inorganic carbon (CO2 + HCO3- + CO32- = dissolved inorganic carbon [DIC]) and subsequently increased to a near-maximum level following photosynthetic depletion of the DIC pool. When DIC accumulation was mediated by the active Na+-dependent HCO3- transport system, the initial rate of fluorescence quenching was found to be highly correlated with the initial rate of H14CO3- transport (r = 0.96), and the extent of fluorescence quenching was correlated with the size of the internal DIC pool (r = 0.99). Na+-dependent HCO3- transport-mediated accumulation of DIC caused fluorescence quenching in either the presence or absence of the CO2 fixation inhibitor glycolaldehyde, indicating that quenching was not due simply to NADP+ reduction. The concentration of Na+ required to attain one-half the maximum rate of H14CO3- transport, at 20 [mu]M external HCO3-, declined from 9 to 1 mM as the external pH increased from 8 to 9.6. A similar pH dependency was observed when fluorescence quenching was used to determine the kinetic constants for HCO3- transport. In cells capable of Na+-dependent HCO3- transport, both the initial rate and extent of fluorescence quenching increased with increasing external HCO3-, saturating at about 150 [mu]M. In contrast Na+-independent HCO3- transport-mediated fluorescence quenching saturated at an HCO3- concentration of about 10 [mu]M. It was concluded that measurement of chlorophyll a fluorescence emission provided a convenient, but indirect, means of following Na+-dependent HCO3- transport and accumulation in Synechococcus.     

Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer

Metallic surfaces can have unusual effects on fluorophores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy transfer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the silver islands so that there are a range of fluorophore-to-metal distances. We show that proximity of fluorophores to the silver islands results in increased fluorescence intensity, with the largest enhancement for the lowest-quantum-yield fluorophores. Importantly, the metal-induced increases in intensity are accompanied by decreased lifetimes and increased photostability. These effects demonstrate that the silver islands have increased the radiative decay rates of the fluorophore. For solvent-sensitive fluorophores the emission spectra shifted to shorted wavelengths in the presence of the silver islands, which is consistent with a decrease of the apparent lifetime for fluorophores near the metal islands. We also observed an increased intensity and blue spectral shift for the protein human glyoxalase, which displays a low quantum yield for its intrinsic tryptophan emission. In this case the blue shift is thought to be due to increased emission from a buried low-quantum-yield tryptophan residue. Increased intensities were also observed for the intrinsic emission of the nucleic acid bases adenine and thymine and for single-stranded 15-mers poly(T) and poly(C). And finally, we observed increased RET for donors and acceptors in solution and when bound to double-helical DNA. These results demonstrate that metallic particles can be used to modify the emission from intrinsic and extrinsic fluorophores in biochemical systems.     

N-Methylaspartate-Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release

The effect of N-methyl-D,L-aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration-dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20-100%. The effect of NMA appeared to be receptor-mediated, as coperfusion with D-2-amino-5-phosphonovaleric acid curtailed the NMA response by some 90%. The NMA-stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist D,L-cis-2,3-piperidine dicarboxylic acid. Although the NMA-evoked release of taurine and PEA was Ca2+-dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA-induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.     

Quenching mechanism of quinolinium-type chloride-sensitive fluorescent indicators

Quinolinium based Cl- sensitive fluorescent indicators have been used extensively to measure intracellular Cl- activity. To define their fluorescence quenching mechanism, a series of N-methyl quinolinium derivatives were synthesized, including N-methylquinolinium (Q), 6-methylQ, 6-methoxyQ, 6-chloroQ, 3-bromoQ, 6-aminoQ and N-methylisoquinolinium. Stern-Volmer plots for quenching by Cl-, Br-, SCN-, I-, F-, OAc- and CO3(2-) from both intensity and lifetime measurements were linear. Bimolecular quenching rate constants (kq) decreased with increasing anion oxidation potentials and increased with increasing quinolinium reduction potentials. The free energy change for charge transfer (deltaG), calculated from indicator spectral and electrochemical properties, was found to correlate with log kq. These results suggest that quenching of quinolinium fluorescence in water by anions involves a charge-transfer quenching mechanism. Understanding the mechanism facilitates structure-based predictions of the anion sensitivities of quinolinium indicators to design improved Cl- indicators with tailored properties.     

Chlorinated hydrocarbon-cell membrane interactions studied by the fluorescence quenching of carbazole-labeled phospholipids: Probe synthesis and characterization of the quenching methodology

In recognition of the need to understand better the interactions of the chlorinated hydrocarbon insecticides with cell membranes we investigated the use of fluorescence quenching of membrane-bound fluorophores by these chlorinated hydrocarbons. An extensive survey of potential fluorophores identified the N-alkyl derivatives of carbazole as being especially suitable fluorophores. The fluorescence emission of these derivatives is quenched by a wide variety of commonly-used chlorinated hydrocarbons. This quenching is collisional and does not result in significant photodecomposition.Four structurally distinct carbazole-labeled phospholipids were synthesized, and their structures were confirmed by 270 MHz proton NMR and by chromatographic and chemical means. The carbazole moiety of each labeled phospholipid should be localized at a different depth in lipid bilayer. However, water soluble quenchers indicate that the fluorophores are inaccessible to the aqueous phase, irrespective of their point of attachment to the phospholipids.When incorporated into lipid bilayers, the fluorescence lifetime of these carbazole-labeled phospholipids reveals the collisional frequency between the fluorophore and the chlorinated hydrocarbon. As a result quenching of membrane-bound fluorophores may be used to measure: (1) the diffusional rate of the chlorinated hydrocarbon in the bilayer; (2) the lipid-water partition coefficient; (3) the maximum binding capacity of the membrane for the chlorinated hydrocarbon. Examples of all these measurements are given, and the fluorometric results are confirmed by direct chemical analysis.     

Mechanism of alcohol‐enhanced lucigenin chemiluminescence in alkaline solution

The chemiluminescence (CL) of lucigenin (Luc²⁺) can be enhanced by different alcohols in alkaline solution. The effect of different fatty alcohols on the CL of lucigenin was related to the carbon chain length and the number of hydroxyl groups. Glycerol provides the greatest enhancement. UV/Vis absorption spectra and fluorescence spectra showed that N‐methylacridone (NMA) was produced in the CL reaction in the presence of different alcohols. The peak of the CL spectrum was located at 470 nm in all cases, indicating that the luminophore was always the excited‐state NMA. The quenching of lucigenin CL by superoxide dismutase (SOD) and the electron spin resonance (ESR) results with the spin trap of 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) demonstrated that superoxide anions (O₂^?–) were generated from dissolved oxygen in the CL reaction and that glycerol and dihydroxyacetone (DHA) can promote O₂^?? production by the reduction of dissolved oxygen in alkaline solution. It was assumed that the enhancement provided by different alcohols was related to the solvent effect and reducing capacity. Glycerol and DHA can also reduce Luc²⁺ into lucigenin cation radicals (Luc^?+), which react with O₂^?? to produce CL, and glycerol can slowly transform into DHA, which is oxidized quickly in alkaline solution. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes

We studied fluorescence intensity, polarization and lifetime of some commonly used fluorophores conjugated to oligodeoxyribonucleotides with different primary and secondary structures. We found that fluorescence intensity can increase or decrease upon hybridization of the labeled strand to its complement depending on the sequence and position of the fluorophore. Up to 10-fold quenching of the fluorescence upon hybridization was observed when the dye moiety was attached close to the 3′ end and the 3′-terminal base was either dG or dC. No quenching upon hybridization was observed when the dye was positioned within the same sequence context but close to the 5′ end. The presence of a dG overhang quenches the fluorescence less efficiently than a blunt end dG-dC or dC-dG base pair. When located internally in the double strand, the dG-dC base pair does not affect the fluorescence of the nearby dye. Guanosine in a single-stranded oligonucleotide quenches the fluorescence of nearby dye by <2-fold. Upon duplex formation, this quenching is eliminated and the fluorescence increases. This increase can only be detected when the fluorophore is located at least 6 nt from the terminal dG-dC base pair. The change of fluorescence polarization upon duplex formation inversely correlates with the change of intensity. Fluorescein conjugated to a single-stranded oligonucleotide or a duplex undergoes a bi-exponential decay with ~4 and ~1 ns lifetimes.     

Phase fluorometric sterilizable optical oxygen sensor

We report here on a low-cost, optical oxygen sensor as an attractive alternative to the widely used amperometric Clark-type oxygen electrode for measuring dissolved oxygen tensions in cell cultures and bioreactor. Our sensor is based on the defferential quenching of the fluorescence lifetime of chromophore in response to the partial pressure of oxygen. This is measured as a phase shift in fluorescence emission from the chromophore due to oxygen quenching when excited by an intensity modulated beam of light. In this article we demonstrate the advantages of lifetime-based optical methods over both intensity based optical methods and amperometric electrodes. Our sensor is particularly suitable for measuring dissolved oxygen in bioreactors. It is autoclavable, is free of maintenance requirements, and solvents the problems of long-term stability, calibration drifts, and reliable measurement of low oxygen tensions in dense microbial cultures that limit the utility of Clark-type elcectordes. (c) 1994 John Wiley & Sons, Inc.     

Multi-path quenchers: efficient quenching of common fluorophores

Fluorescence quenching groups are widely employed in biological detection, sensing, and imaging. To date, a relatively small number of such groups are in common use. Perhaps the most commonly used quencher, dabcyl, has limited efficiency with a broad range of fluorophores. Here, we describe a molecular approach to improve the efficiency of quenchers by increasing their electronic complexity. Multi-Path Quenchers (MPQ) are designed to have multiple donor or acceptor groups in their structure, allowing for a multiplicity of conjugation pathways of varied length. This has the effect of broadening the absorption spectrum, which in turn can increase quenching efficiency and versatility. Six such MPQ derivatives are synthesized and tested for quenching efficiency in a DNA hybridization context. Duplexes placing quenchers and fluorophores within contact distance or beyond this distance are used to measure quenching via contact or FRET mechanisms. Results show that several of the quenchers are considerably more efficient than dabcyl at quenching a wider range of common fluorophores, and two quench fluorescein and TAMRA as well as or better than a Black Hole Quencher.     

Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO(2)-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO(2) conditions. Ethoxyzolamide (0.4 mM), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mM acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to beta-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO(2)-binding site appeared to be unique among the beta-CAs so far reported.     

Fluorescence lifetime characterization of novel low-pH probes.

The structures and functions of the cellular acidic compartments are strongly dependent on the pH gradients across vesicular membranes. Measurement and imaging of the vesicular pH require fluorophores with appropriate pK(a) values. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulness to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases that selectively accumulate in acidic vesicles after being protonated. They have higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffer pH increased the quenching effects of their basic side chains and substantially reduced their steady-state fluorescence and lifetimes. The apparent pK(a) values determined from their lifetime responses were shifted to near neutral values because of the dominant intensity contribution from their protonated species. One unique property of LysoSensor DND-189 is its nonmonotonic lifetime responses of the maxima occurring between pH 4 and 5. LysoSensor DND-192 did not show significant lifetime changes over a wide pH range. LysoSensor DND-160, which was the only excitation and emission ratiometric probe, showed significant pH-dependent lifetime changes as well as its spectral shifts. Its apparent pK(a) values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Because of the pH-dependent absorption spectra, the apparent pK(a) values could be manipulated between 3 and 5 by changing the excitation and/or emission wavelengths. These results indicate that LysoSensor DND-160 is a promising probe for lifetime imaging to determine lysosomal pH.     

Solvent accessibility of the adenosine 5'-triphosphate catalytic site of sarcoplasmic reticulum CaATPase

The CaATPase of rabbit skeletal sarcoplasmic reticulum was labeled at or near the ATP catalytic site with fluoresceinyl isothiocyanate (FITC), and the accessibility of the attached probe to the bulk solvent was determined by I- quenching of its fluorescence. The quenching of free FITC was also measured. In both cases, the quenching was of the Stern-Volmer type and collisional quenching rate constants were obtained over the pH range 5-8 in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and with added Ca2+, vanadate, or phosphate. The fluorescence intensity and susceptibility to quenching by I- of free FITC were insensitive to the added ligands. In all cases, the intensity decreased with pH, as predicted from the known properties of FITC mono- and dianions. The collisional quenching rate constants increased at lower pH, as expected for I- quenching of a molecule with decreasing negative charge due to protonation. When FITC was attached to the CaATPase, the FITC fluorescence intensity and I- collisional quenching rate constants were sensitive to ligand binding as well as pH. The changes in fluorescence intensity with acidity, when compared to the results for free FITC, indicated the pKa of the FITC was reduced 0.6 unit when it was attached to the CaATPase. Excited-state lifetime measurements indicated that ligand effects at constant pH were not due to protonation-induced changes in FITC quantum yield but to conformational changes of the CaATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein's binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (alpha-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4'R,8'R-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.     

Effect of N-methyl-d,l-aspartate on luteinizing hormone secretion in ovariectomized ewes in the absence and presence of estradiol

N-methyl-d,l-aspartate (NMA), a potent agonist of the neuroexcitatory amino acids aspartate and glutamate, stimulates release of luteinizing hormone (LH) in rats and nonhuman primates. The objective of the experiments described here was to determine the effect of NMA on LH secretion in ovariectomized ewes, in both the absence and presence of estradiol. In Experiment 1, blood samples were collected from 16 ewes every 12 min for 4 h. At Hour 2, ewes received i.v. injections of either 0, 6, 12, or 24 mg NMA/kg body weight dissolved in 0.9% saline (n = 4 per treatment). Mean LH concentrations were unaltered by any dose of NMA (p greater than 0.3). Immediately after completion of Experiment 1, each ewe received an s.c. Silastic implant designed to maintain circulating concentrations of estradiol of approximately 1 pg/ml. Three weeks later, Experiment 2 was conducted, using the same blood sampling regimen and doses of NMA as Experiment 1. The estradiol implants decreased serum LH concentrations in all animals. Treatment with saline failed to alter mean LH concentrations (p greater than 0.3). In contrast, 6, 12, and 24 mg NMA/kg body weight increased mean LH concentrations by 326% (p less than 0.03), 1125% (p less than 0.02), and 441% (p less than 0.0001), respectively. These results demonstrate that exogenous estradiol suppresses LH release in sheep in a manner antagonized by NMA.     

CO2 fixation and its regulation in Anacystis nidulans (Synechococcus)

Anacystis nidulans (Synechococcus) had a minimal doubling time of 5 hrs at 30 degrees C at saturating light intensity and carbon dioxide concentration. Half maximal growth rates in saturating CO2 occured at a light intensity of 0.54 mW per cm2, and there was an apparent threshold intensity of 0.13 mW per cm2 below which no growth occurred. Growth rate in saturating light was dependent on the concentration of CO2+H2CO3 in the medium, rather than on total dissolved CO2; half maximal rates were estimated at 0.1 mM CO2+H2CO3. Under saturating conditions of light and CO2, 14CO2 was fixed primarily into 3-PGA, and subsequently moved into sugar phosphates and amino acids. Incorporation into aspartate was relatively slow. CO2 fixation was strictly light-dependent. The changes in adenylate and pyridine nucleotide pools were followed in light/dark and dark/light transitions. Whereas adenylates relaxed slowly over 15-20 min to the concentrations characteristic of illuminated cells following the abrupt changes induced by darkening, the sharp drop in intracellular NADPH showed little dark recovery although rapid restoration occurred on reillumination. Other pyridine nucleotides showed no changes during these transitions. The nucleotide specificity and Km of partially purfied GAP dehydrogenase suggest a role for this enzyme in the regulation of CO2 fixation.     

Novel trifunctional carrier molecule for the fluorescent labeling of haptens

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)     

温度和CO2浓度升高下转Bt水稻种植对土壤活性碳氮和线虫群落的短期影响

全球气候变化下转Bt水稻种植对土壤生态系统的影响仍是未知领域。利用野外开顶式气室(OTC)模拟气候变化,探讨了温度和CO2浓度([CO2])升高后种植转Bt(华恢1号)水稻对土壤活性碳氮和线虫群落的影响。结果表明:(1)温度和[CO2]升高条件下种植转Bt水稻显著影响土壤可溶性碳(DOC)、可溶性氮(DON)和硝态氮(NO-3-N)含量,同时DOC和DON受到升温和[CO2]升高与转Bt水稻的交互影响;在正常温度和[CO2]下,转Bt水稻显著降低了土壤微生物量碳氮含量(MBC、MBN),但在温度和[CO2]升高后转Bt水稻种植使土壤MBC和MBN含量显著高于亲本水稻。(2)在温度和[CO2]升高条件下,转Bt水稻土壤线虫总数呈高于亲本水稻的趋势,植食性线虫的比例随[CO2]和温度升高呈增大的趋势。此外,升温和转Bt水稻种植提高了土壤线虫群落的能流通道指数,而[CO2]升高和转Bt水稻种植则提高了土壤线虫群落的富集指数。总之,在模拟全球气候变化下,种植转Bt水稻在短期内对土壤活性碳氮和线虫群落均产生影响,但短期研究并未发现转Bt水稻种植对土壤生态系统的不利影响。     

Lifetime and temporal occurrence of ectomycorrhizae on ponderosa pine (Pinus ponderosa Laws.) seedlings grown under varied atmospheric CO2 and nitrogen levels

Climate change (elevated atmospheric CO2, and altered air temperatures, precipitation amounts and seasonal patterns) may affect ecosystem processes by altering carbon allocation in plants, and carbon flux from plants to soil. Mycorrhizal fungi, as carbon sinks, are among the first soil biota to receive carbon from plants, and thereby influence carbon release from plants to soil. One step in this carbon release is via fine root and mycorrhizal turnover. It is necessary to know the lifetime and temporal occurrence of roots and mycorrhizae to determine the capacity of the soil ecosystem to sequester carbon assimilated aboveground. In this study, ponderosa pine (Pinus ponderosa Laws) seedlings were grown under three levels of atmospheric CO2 (ambient, 525 and 700 mol CO2 mol-1) and three levels of annual nitrogen additions (0,100 and 200 kg N ha-1) in open-top chambers. At a two-month frequency during 18 months, we observed ectomycorrhizal root tips observed using minirhizotron tubes and camera. The numbers of new mycorrhizal root tips, the numbers of tips that disappeared between two consecutive recording events, and the standing crop of tips at each event were determined. There were more mycorrhizal tips of all three types seen during the summer compared with other times of the year. When only the standing crop of mycorrhizal tips was considered, effects of the CO2 and N addition treatments on carbon allocation to mycorrhizal tips was weakly evident. However, when the three types of tips were considered collectively, tips numbers flux of carbon through mycorrhizae was greatest in the: (1) high CO2 treatment compared with the other CO2 treatments, and (2) intermediate N addition treatment compared with the other N addition treatments. A survival analysis on the entire 18 month cohort of tips was done to calculate the median lifetime of the mycorrhizal root tips. Average median lifetime of the mycorrhizal tips was 139 days and was not affected by nitrogen and CO2 treatments.     

Fluorescence quenching studies of nitrated polycyclic aromatic hydrocarbons

The analysis of nitrated polycyclic aromatic hydrocarbons (NPAHs) is of great importance because of the mutagenicity and possible carcinogenic activity of these compounds, which are distributed widely in the environment. Nitro‐substituents in aromatic compounds are known to quench fluorescence and NPAHs have no intrinsic fluorescence, but they can be determined using their quenching effects on other fluorophores. The quenching effects of several important NPAHs on 1,2,3,4‐ tetrahydro‐1‐naphthol,5,6,7,8‐tetrahydro‐1‐naphthol,4‐(2‐hydroxy‐4‐sulfo‐1‐naphthylazo)‐2‐naphthalene carboxylic acid and 7‐amino‐4‐methyl coumarin have been studied. The singlet emission of these fluorophores is efficiently quenched by all the NPAHs, the quenching following the Stern–Volmer relationship. Quenching constants and the limits of detection and linear ranges of the quenchers have been determined in each case: the limits of detection are ca 1 µm . Copyright © 2010 John Wiley & Son, Ltd.     

Calibration of the parallax fluorescence quenching method for determination of membrane penetration depth: refinement and comparison of quenching by spin-labeled and brominated lipids.

We previously introduced the "parallax" method, which uses fluorescence quenching by spin-labeled lipids in order to measure the depth of molecules within a membrane [Chattopadhyay, A., & London, E. (1987) Biochemistry 26, 39-45]. In this report the accuracy of this method is established by comparison of spin-label quenching to that obtained using brominated lipids. To accomplish this, the fluorescent molecules used were a fatty acid labeled with a carbazole buried deeply within the acyl chain region of the membrane, an acyl-Trp with the Trp residue residing near the polar membrane region, and cytochrome b5, which has Trp residues in its membrane-inserted region. The depths calculated from the amount of bromine quenching agreed with those determined using parallax analysis. This indicates that the depth reported by parallax analysis is accurate and that the spin labels residue very close to their predicted locations in the membrane. Furthermore, there was good agreement when parallax analysis was applied both to quenching by brominated and spin-labeled molecules, suggesting that the analysis is valid in both cases. The effect that different distributions and motions of fluorophores and quenchers would have on parallax analysis was also examined. For uniform distributions of quenchers or fluorophores over a range of depths, it was found that the analysis reports the average fluorophore depth. In addition, experimental data suggest that motional effects do not significantly alter the measured depths. This is consistent with the motions during the short excited state lifetime of the fluorophores being relatively small and/or relatively isotropic.