Validity of Using Background Leukemia Incidence Rates with Cohort Mortality-Based Potency Estimates to Calculate Excess Lifetime Risk

Data from occupational cohort mortality studies have been used to derive exposure-response curves and general population excess lifetime cancer risks, given low-level, chronic exposure. Using an actuarial method, mortality-based rate ratios associated with cumulative exposures are applied to age-specific background cancer mortality rates for a theoretical population aged birth to 70 years. In one recent U.S. Environmental Protection Agency health assessment, a mortality-based leukemia relative rate model was used with background leukemia incidence rates, rather than mortality rates, to calculate excess lifetime risk of leukemia incidence. We examined the validity and implications of this novel approach, while considering possible bias if a potential leukemogen did not pose equal risk by cell type. Limited sensitivity analyses were also conducted. Our analyses show that using total leukemia mortality-based potency estimates with background incidence rates will introduce a biased estimate of excess lifetime risk, the direction of which varies by potency and the histological type of leukemia. These biases were somewhat increased on adjustment for possible greater susceptibility of children. For potent carcinogens, the traditional approach provides a reasonable approximation of excess lifetime mortality risk for both the more and less fatal forms of leukemia, even after adjustments for children and is, therefore, to be preferred. Less consistency by leukemia cell type and background rate was observed for flatter exposure-response curves. This evaluation illustrates the importance of carefully examining the impact of methodological changes to calculations of excess lifetime risk before implementation.     

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5′-GMP, ribose-5-phosphate, ketone bodies, and purines.     

The influence of dead time related distortions on live cell fluorescence lifetime imaging (FLIM) experiments

Recent developments in the field of fluorescence lifetime imaging microscopy (FLIM) techniques allow the use of high repetition rate light sources in live cell experiments. For light sources with a repetition rate of 20–100 MHz, the time‐correlated single photon counting (TCSPC) FLIM systems suffer serious dead time related distortions, known as “inter‐pulse pile‐up”. The objective of this paper is to present a new method to quantify the level of signal distortion in TCSPC FLIM experiments, in order to determine the most efficient laser repetition rate for different FLT ranges. Optimization of the F ‐value, which is the relation between the relative standard deviation (RSD) in the measured FLT to the RSD in the measured fluorescence intensity (FI), allows quantification of the level of FI signal distortion, as well as determination of the correct FLT of the measurement. It is shown that by using a very high repetition rate (80 MHz) for samples characterized by high real FLT's (4–5 ns), virtual short FLT components are added to the FLT histogram while a F ‐value that is higher than 1 is obtained. For samples characterized with short real FLT's, virtual long FLT components are added to the FLT histogram with the lower repetition rate (20–50 MHz), while by using a higher repetition rate (80 MHz) the “inter‐pulse pile‐up” is eliminated as the F ‐value is close to 1. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Picosecond fluorescence kinetics and energy transfer in chloroplasts and algae

Single-photon timing with picosecond resolution is used to investigate the kinetics of the fluorescence emission of chlorophyll a in chloroplasts from spinach and pea and in the algae Chlorella pyrenoidosa and Chlamydomonas reinhardii. The fluorescence decay is best described by three exponential components in all species. At low light intensity and with open reaction centers of Photosystem II (F₀), we find lifetimes of approx. 100, 400 and 1100 ps for the three components. Closing the reaction centers by addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea plus hydroxylamine and by increasing light intensity produces only minor changes in the almost constant fast- and medium-lifetime components; however, there is a dramatic increase in the yield of the slow component, by a factor of about 20, accompanied by only a modest increase in the lifetime to 2200 ps (F_max). In good agreement with previous fluorescence lifetime measurements, we find an increase in the averaged lifetime of the three components from 0.5 to 2.0 ns, which is proportional to the 4-fold increase in the total fluorescence yield. Our time-resolved results are inconsistent with models which are based on the proportionality between lifetime and yield and which involve a homogeneous origin of fluorescence that is sensitive to the state of the reaction centers. We conclude that the variable part of the fluorescence, which is dominated by the slow phase, reflects the kinetics of charge recombination in the reaction center, as proposed previously (Klimov, V.V., Allakhverdiev, S.I. and Paschenko, V.Z. (1978) Dokl. Akad. Nauk S.S.S.R. 242, 1204–1207). The modest increase in lifetime of the slow phase indicates the presence of some energy transfer between photosynthetic units.     

Fluorescence decay studies of modified dinucleoside monophosphates containing 1-N6-ethenoadenosine

Five dinucleoside monophosphates containing I-N⁶-ethenoadenosine (?A) have been studied using fluorescence measurements. The fluorescence spectra of these dinucleoside monophosphates are almost the same as the fluorescence spectrum of ?AMP. Fluorescence quantum yields of these dimers are greatly reduced compared to that of ?AMP. Intramolecular base-base interactions may be responsible for fluorescence quenching. It is found that the fluorescence decay kinetics does not obey a simple decay law but that the decay data can be well described as a sum of three exponentials. This implies that these dimers cannot be characterized as a two-state system, but can be described as systems consisting of three or more conformational states. Sequence effects upon the fluorescence behavior are observed. The fluorescence quenching and decay parameters of Gp?A and Up?A indicate a higher degree of base-base interaction than in their ?ApG and ?ApU counterparts.     

Lifetimes of Arabidopsis cryptochrome signaling states in vivo

One crucial component in light signaling is the quantity of photoreceptor present in the active signaling state. The lifetime of the signaling state of a photoreceptor is limited because of thermal or otherwise back reversion of the chromophore to the ground state, and/or degradation of the photoreceptor in the light‐activated state. It was previously shown that the lit state of plant cryptochromes contains flavin‐neutral semiquinone, and that the half‐lives of the lit state were in the range of 3–4 min in vitro. However, it was unknown how long‐lived the signaling states of plant cryptochromes are in situ. Based on the loss of degradation of cry2 after prolonged dark incubation and loss of reversibility of photoactivated cry1 by a pulse of green light, we estimate the in vivo half‐lives of the signaling states of cry1 and cry2 to be in the range of 5 and 16 min, respectively. Based on electron paramagnetic resonance measurements, the lifetime of the Arabidopsis cry1 lit state in insect cells was found to be ~6 min, and thus very similar to the lifetime of the signaling state in planta. Thus, the signaling state lifetimes of plant cryptochromes are not, or are only moderately, stabilized in planta.     

Fluorescence spectroscopic studies of pyrene-actin adducts

Reaction kinetic studies of the sulfhydryl-directed fluorescent probes N-(1-pyrene)maleimide (PM) and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle showed that there were three accessible sulfhydryl groups in actin. Fluorescence spectral studies showed energy transfer from aromatic amino acid residues to fluorophore reacted at Cys-373, as well as weak excimer fluorescence probably due to doubly labeled molecules at Cys-10 and Cys-373. These results provide further evidence that trytophan and tyrosine residues are located near the probe attached to Cys-373 or Cys-10 and the latter two thiols are in close proximity. In aged PM-Iabeled F-actin, the succinimido ring of PM underwent intramolecular aminolysis. resulting in large emission spectral changes and increased excimer fluorescence. Solvent perturbation studies indicate that the probes were located in a hydrophobic environment; their quantum yield and spectrum properties were very sensitive to changes in the microenvironment. Nanosecond-pulse fluorimetry studies revealed complex fluorescence emission decays with three intrinsic lifetimes in adducts with low molecular weight thiols as well as in labeled proteins. Fluorescence lifetimes were 17. 48 and 111 ns for the pyrenemaleimide adduct of actin, and 3, 14 and 60 ns for the pyrenyliodoacetamide adduct. Supporting evidence is given for the argument that multiple fluorescence lifetimes are an intrinsic property of the pyrene derivatives and are not due to the presence of impurity or heterogeneity in the protein reaction sites. Because of their high sensitivity and long lifetimes, pyrene derivatives are extremely useful.     

Examination of Phycomyces blakesleeanus for nitrate reductase as a possible blue light photoreceptor

Phycomyces blakesleeanus is unable to grow on media which contain nitrate as the sole nitrogen source. Further, according to a number of assay procedures, there is no significant nitrate reductase activity in Phycomyces. Thus, although nitrate reductase has been proposed to be a blue-light receptor in Neurospora, no active nitrate reductase is available to serve this function in Phycomyces.