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Flash-induced voltage changes in halorhodopsin from Natronobacterium pharaonis.

The flash-induced voltage response of halorhodopsin at high NaCl concentration comprises two main kinetic components. The first component with tau approximately 1 micros does not exceed 4% of the overall response amplitude and is probably associated with the formation of the L (hR520) intermediate. The second main component with tau approximately 1-2.5 ms which is independent of Cl- concentration can be ascribed to the transmembrane Cl- translocation during the L intermediate decay. The photoelectric response in the absence of Cl- has the opposite polarity and does not exceed 6% of the overall response amplitude at high NaCl concentration. A pH decrease results in substitution of the Cl(-)-dependent components by the photoresponse which is similar to that in the absence of Cl-. Thus, the difference between photoresponses of chloride-binding and chloride-free halorhodopsin forms resembles that of bacteriorhodopsin purple neutral and blue acid forms, respectively. The photovoltage data obtained can hardly be explained within the framework of the photocycle scheme suggested by Varo et al. [Biochemistry 34 (1995), 14490-14499]. We suppose that the O-type intermediate belongs to some form of halorhodopsin incapable of Cl- transport.

Bacterial Proteins↗

Automated normal-phase preparative high-performance liquid chromatography as a substitute for flash chromatography in the synthetic research laboratory.

An automated normal-phase preparative HPLC system was developed in order to omit time-consuming flash column chromatography in the synthetic research laboratory. The system is equipped with steel columns packed with spherical 12 microm silica and is able to separate samples in a range of 0.1-10 g depending on the column diameter and chromatographic problem. It was designed to be used as an open access instrument in the research department. The general users select from binary gradient programs after running an analytical TLC with the raw product. The HPLC instrument was fully controlled by the Chromeleon software from Dionex. A Gilson 215 robot served as injector/collector.

Automation↗

Gas chromatographic studies of phenobarbital and diphenylhydantoin after flash-heater alkylation.

The functionally excellent flash-heater methylation, ethylation and butylation techniques have been extended to include the higher alkyl homologs. Phenobarbital and diphenylhydantoin have been alkylated using the tetraalkylammonium hydroxides in which the alkyl group ranges from methyl to hexyl. The gas chromatographic properties of the resulting alkyl derivatives have been investigated.

Alkylation↗

Simple off-line flash pyrolysis procedure with in situ silylation for the analysis of hydroxybenzenes in humic acids and coals.

A simple device consisting of a glass pyrolysis chamber fitted for a commercial resistively heated pyrolysis probe and connected to a solvent desorption tube for air monitoring was applied to off-line pyrolysis under silylating conditions of humic acids (soil, lake) and coals. Samples were flash pyrolysed at 700 degrees C in the presence of excess hexamethyldisilazane, and evolved products were swept off by a nitrogen stream and trapped onto a charcoal filter from where they were desorbed with dichloromethane and analysed by gas chromatography (GC)-mass spectrometry. Humic acids afforded trimethylsilyl (TMS) ethers of phenols, 2-methoxyphenols (guaiacols), 2,6-dimethoxyphenols (syringols), and dihydroxy and trihydroxybenzenes as major products. TMSoxy benzenes were the principal products observed from pyrolysis/silylation of coals. In comparison with conventional pyrolysis, the in-situ derivatisation process enhances the levels of phenols with respect to hydrocarbons and improves the GC separation of isomers (e.g. meta- from para-cresol). With respect to tetramethylammonium hydroxide thermochemolysis, pyrolysis/silylation operates under milder conditions and permits discrimination between free and methylated hydroxy groups. The performance of the method for the quantitative determination of evolved product is described. Yields of evolved silylated mono and dihydroxybenzenes occur in the mg/g range with relative standard deviations generally between 16 and 30%.

Coal↗

Glass capillary column gas chromatography of barbiturates after flash-heater derivatization with dimethylformamide dimethylacetal.

Glass capillary column gas chromatography has been used for quantitative and qualitative analyses of barbiturates. The barbiturates are first converted into the corresponding acetal derivates by flash-heater derivatization, using dimethylformamide dimethylacetal as the derivatization reagent. The influence of the injection port temperature, the column temperature, the solvent used to dissolve the substances and the reagent dilution were studied in order to obtain the optimal response with good reproducibility. Calibration graphs in the concentration range 5--50 micrograms/ml were evaluated and the relative standard deviations for the quantitative analyses were calculated. High sensitivity was obtained when extracts from plasma containing phenobarbital in the low therapeutic concentration range were analysed using the described derivatization technique.

Barbiturates↗

A flash-type bioluminescent immunoassay that is more sensitive than radioimaging: quantitative detection of cytokine cDNA in activated and resting human cells.

Because of its high sensitivity, bioluminescence (BL) is an excellent alternative to radioactive quantitation of cytokine RT-PCR-derived products. BL also allows detection of amplicons at cycle numbers not normally detectable using radioactivity. No direct comparisons between these two methods have been made. In this study, the sensitivities of BL using recombinant aequorin, a flash-type luminescent tag capable of detecting signal to attomolar (10(-18) M) levels and radio imaging (RI) were directly compared. In addition, the application of BL for detecting cytokine message from biologic samples was examined. BL was 30- to 60-fold more sensitive than RI in detecting human IL-2 and CD3delta amplicons. This difference was particularly found during low cycle PCR, but was less at higher cycle numbers. The ability of BL to detect differences in cytokine message in stimulated and unstimulated human peripheral blood mononuclear cells was also evaluated. Using linear regression analysis, we observed up to 5,000-fold increases in RT-PCR amplified-mRNA in stimulated cells for IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10 and GM-CSF compared to unstimulated cells. Changes in CD3delta, TNF alpha or IL-12 were not observed or quantitated. We present a novel aequorin-based application of bioluminescent technology to directly quantitate RT-PCR amplicons and to investigate the induction of human cytokine expression. Significant advantages of this sensitive bioluminescent method compared with radioactive methods are its abilities to quantitate amplicons in a PCR cycle range where linear detection is most robust and to analyze products in an automated, open-architecture microtiter plate format.

Aequorin↗

Auditory and visual event-related potentials and flash visual evoked potentials in Alzheimer's disease: correlations with Mini-Mental State Examination and Raven's Coloured Progressive Matrices.

We investigated possible correlations among neurophysiological examinations [auditory and visual event-related potentials (A-ERPs, V-ERPs), and flash visual evoked potentials (F-VEPs)] and neuropsychological tests [Mini-Mental State Examination (MMSE) and Raven's Coloured Progressive Matrices (RCPM)] in 15 subjects with probable or possible Alzheimer's disease (AD) according to the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) criteria. The P300 latency of A-ERPs was correlated with the scores of MMSE but not with those of RCPM. The P300 latency of V-ERPs was more significantly correlated with the scores of RCPM than with those of MMSE. The P2 latency of F-VEPs was more significantly correlated with the scores of RCPM than with those of MMSE. The P2 latency of F-VEPs was not correlated with the P300 latency of A-ERPs but was correlated with the P300 latency of V-ERPs. The close relationship among V-ERPs, F-VEPs and RCPM suggests that these examinations at least partly reflect the functions of visual association areas in AD. Furthermore, discrepancy between P300 latency by A-ERPs and V-ERPs suggests that the mechanism responsible for P300 generation is not identical between these two stimulus modalities.

Aged↗

Evaluation of different recording parameters to establish a standard for flash electroretinography in rodents.

Different electrodes and stimulus protocols commonly used for electroretinography in rodent eyes were compared for convenience of use, degree of damage to corneal epithelium, and for magnitude of amplitude, reproducibility, left versus right eye accuracy, and reliability of recorded parameters of the flash electroretinogram (ERG). Adult C57BL/6 pigmented mice and albino Wistar rats were used to determine scotopic ERGs in response to Ganzfeld or strobe-light stimulation and light-adapted (photopic) ERGs recorded from both eyes at the same time. Test-retest data were used for statistical analyses to compare a monopolar gold-wire contact lens electrode (CLE), a cotton-wick silver-silver chloride electrode (CSCE), a DTL fiber electrode (DTLE), and a circular stainless steel wire electrode (SSE). Corneas were evaluated for abrasion after ERG recordings using fluorescein staining and also for the time taken, ease of insertion, and re-insertions required for the different electrodes. Compared to CSCE, DTLE, and SSE, the ERG potentials recorded by CLE had significantly larger scotopic amplitudes and oscillatory potentials under strobe or Ganzfeld stimulation and for light-adapted ERG b-wave amplitudes in both mice and rats. In analyzing test-retest data of scotopic ERG a-wave and b-wave amplitudes, the intraclass correlation coefficient showed the best agreement for the CLE (range 0.61-0.94) compared to the SSE (0.13-0.77), DTLE (0.02-0.69), and CSCE (0.12-0.51). In mice and rats, logistic regression analyses revealed significant correlations for amplitudes of most scotopic ERG parameters between contralateral eyes obtained with CLE and for some ERG components recorded by SSE. When comparing ERG amplitudes for stimulation by strobe or Ganzfeld, the difference was least with the CLE compared to DTLE, CSCE, or SSE. The time taken to insert the four different electrodes was greatest for the CLE in both mice and rats. The extent of corneal abrasion resulting from electrode use in mice was largest for the SSE followed by the CLE. However, in rats there was almost no corneal damage after ERG recordings with the CLE. Because of the stability of eye contact, the CLE allows ERGs to be determined over a longer recording session. Recording of scotopic and photopic (light-adapted) ERGs in rodents with monopolar gold-wire contact lens electrodes provides greater amplitudes and higher reproducibility when compared to other commonly used corneal electrodes. These electrodes are significantly better overall than others that were evaluated and should be considered for a standard protocol to monitor retinal function in rodent eyes.

Analysis of Variance↗

The effect of photon noise on the detection of white flashes.

Thresholds for detecting brief, white, foveal test flashes drop abruptly within 0.2 sec of the offset of a white adapting field. The magnitude of the abrupt drop is proportional to the square root of field intensity (square root of I) correct for bleaching and dark light. Thresholds are then stable out to 1.6 sec for 200 msec tests, or recover only slightly for 20 msec tests. These results exclude some simple deterministic models in which Weber-like gain controls in the luminance pathway are assumed to recover exponentially in the dark, but can be explained parsimoniously if turning off the field abolishes photon-driven noise, improving the S/N ratio while leaving visual responsivity virtually unaltered. This theory was first put forward by Krauskopf and Reeves [(1980) Vision Research, 20, 193-196] for S-cone thresholds; it implies that the Weber law for increment thresholds is not due to a single gain control, but rather expresses the product of two distinct square root of I factors, adjustment of responsivity and photon-driven noise. Removal of the noise, not recovery of gain, permits thresholds to fall in early dark adaptation.

Adaptation, Ocular↗

Temporal analysis of the chromatic flash VEP--separate colour and luminance contrast components.

Temporal analysis of the chromatic flash visual evoked potential (VEP) was studied in human subjects with normal and anomalous colour vision using a deterministic pseudo-random binary stimulus (VERIS). Five experiments were carried out on four normal subjects investigating heterochromatic red-green exchange and single colour/achromatic (either red/grey or green/grey) exchange over a wide range of luminance ratios for the two stimuli, the effects of lowered mean luminance on the chromatic VEP and the effects of colour desaturation at constant mean luminance and constant luminance contrast. Finally, the performance of three dichromats, a protanope and two deuteranopes, on heterochromatic exchange VEP and on colour desaturation were investigated. In contrast to the chromatic electroretinogram, which shows great symmetry with respect to luminance ratio on opposite sides of the isoluminant point, the chromatic VEP demonstrated a distinct asymmetry when the colours exchanged included red. On the red side of isoluminance (red more luminant than green), a wave with longer latency and altered waveform became dominant. The effects of green stimulation were indistinguishable from those of achromatic stimulation at the same luminance contrast over the whole range of chromatic contrast and for all levels of desaturation studied. Desaturation of red with constant luminance contrast (desaturated red/grey stimulation) resulted in a systematic alteration in the evoked waveform. Subtraction of the achromatic first- and second-order responses from responses recorded in the red desaturation series resulted in remarkably uniform waveforms, with peak amplitudes growing linearly with saturation. The absence of interaction between achromatic and coloured components for all (including the most intense colour) stimulus parameters used suggests that the generators of these components are separate. Recordings from the dichromats showed that the contrast response minimum shifted from the point of photopic isoluminance to the point of zero cone contrast (at the silent substitution point) for the remaining cone type. The waveforms recorded with a series of luminance ratios were much simpler than those recorded from trichromats and symmetrical with respect to their isoluminant points. Despite the indication of the presence of L cones of apparently normal spectral sensitivity in the deuteranopes (on the basis of flicker photometry), there was no evidence for a red-sensitive component in the desaturation or heterochromatic stimulation series. The results are discussed in terms of the possibility of separate generation of chromatic and achromatic contributions to the VEP.

Color Perception↗

S-cone contribution to pupillary responses evoked by chromatic flash offset.

On a green or red background, the action spectrum of the pupillary responses evoked following the offset of chromatic test flashes shows a prominent short-wavelength lobe and suggests the contribution from photoreceptors other than the previously inferred M- and L-cones (Kimura & Young, Vision Research (1996). 36, 1543-1550), most likely from S-cones. Systematic changes in the shape of the intensity versus amplitude functions with test wavelengths and in the shape of the short-wavelength lobe with response amplitude criteria suggest an antagonistic interaction involving the short- and longer-wavelength photoreceptors.

Color Perception↗

Contribution of hypothermia to effects of chloral hydrate on flash evoked potentials of hooded rats.

This study examined the contribution of hypothermia to the effects of chloral hydrate on the flash evoked potential (FEP) of hooded rats. Three experiments were performed, all employing intraperitoneal injections of saline, and of 75, 150 and 300 mg chloral hydrate/kg body weight. In the first experiment, body temperature was measured in a standard (23 degrees C) environment for 6 hr following injection. Rats were hypothermic following administration of the 150 and 300 mg/kg dosages for up to 1 and 2 hr, respectively. In the second experiment, FEPs were recorded from the visual cortex of chronically implanted rats 30 min after injection (22 degrees C environment). P1N1, N1P2 and P2N2 amplitudes and P1, N1, P2, N2 and P3 peak latencies were significantly increased by the 300 mg/kg dosage. Increased latencies were also noted for the primary components with the 150 mg/kg dosage. The final experiment replicated the second experiment, but at an ambient temperature of 30 degrees C, which prevented hypothermia. Amplitudes were unaffected by chloral hydrate. Significantly increased peak latencies were observed, even with the 75 mg/kg dose for some components. However, the magnitude of the latency increases of the primary components was less than half of that found with a standard environment. These results indicate that depending upon ambient temperature, hypothermia may contribute to chloral hydrate-induced alterations in FEPs.

Animals↗

Ketamine alters rat flash evoked potentials.

Discovering the neurotransmitters involved in the generation of flash evoked potentials (FEPs) would enhance the use of FEPs in screening for and assessment of neurological damage. Recent evidence suggests that the excitatory amino acids, glutamate and aspartate, may be transmitters in the visual system. Ketamine selectively antagonizes the actions of excitatory amino acids on n-methyl-d-aspartate receptors and may be administered systemically. Two experiments were designed to test the effects of ketamine on rat FEPs. First, the effects of ketamine (37, 75, 150 mg/kg) on FEPs recorded in light and dark backgrounds were investigated at a single (10 min) posttreatment interval. Ketamine administration resulted in dose-dependent alterations in FEP peak amplitudes and latencies. Peak P1 amplitude increased by a factor of 4, in a dose-dependent manner. Peak N1 virtually disappeared at 150 mg/kg. Peak P2 amplitude increased by 50%, but only in the light background, and only at 150 mg/kg. Second, ketamine (150 mg/kg) effects on FEPs were investigated 5 min and 30 min following administration. The decrease in peak N1 amplitude was maximal 5 min after administration and the amplitude was recovering at 30 min. The effects on peak P1 and peak N3 amplitudes were maximal 5 min after ketamine administration, but were not recovering 30 min postinjection. The various peak latencies were also affected differently. The possible role of glutamate or aspartate in the generation of rat FEPs is discussed.

Animals↗

THIP, a selective gamma-aminobutyric acid receptor agonist, alters flash-evoked potentials in rats.

This study examined the effects of the GABA(A) agonist THIP on flash-evoked potentials (FEPs) recorded from the primary visual cortex (VC) and superior colliculus (SC) of chronically implanted hooded rats. Animals were given I.P. injections of saline, and of 8, 16, and 24 mg THIP/kg body weight on separate days. Evoked potentials were recorded at 5, 20, and 35 min following injection. Animals were tested at a standard (22.5 degrees C) room temperature. Most significant effects were observed at the 20- and 35-min recording intervals for both the 16 and 24 mg/kg doses, with effects at the 24 mg/kg dose the most pronounced. VC P1 amplitude remained unchanged, while N1 was reduced to such an extent that it became positive, ultimately blending into the rising phase of a positive component appearing between N1 and P2. This positive component had a latency of about 6 ms longer than N1, and became larger in amplitude than P1 at the 24 mg/kg dose. P2 amplitude was drastically reduced, becoming negative. In contrast, components N2 and P3 were augmented, while the amplitude of N3 was unchanged. In the SC, P1 was augmented while P3 was reduced in amplitude. A biphasic (increase/decrease) effect was observed in the N4 complex. In both the VC and SC, latencies of most components were increased, with the late components in the VC increased to the greatest extent. A mild hypothermia was observed at 16 and 24 mg/kg. The results suggest that the GABA(A) receptor plays an important role in the elaboration of the middle (N1-P2) components of FEPs recorded from the rat VC, and that GABAergic mechanisms can influence other components in the VC and SC as well.

Animals↗

Eye light flashes on the Mir space station.

The phenomenon of light flashes (LF) in eyes for people in space has been investigated onboard Mir. Data on particles hitting the eye have been collected with the SilEye detectors, and correlated with human observations. It is found that a nucleus in the radiation environment of Mir has roughly a 1% probability to cause an LF, whereas the proton probability is almost three orders of magnitude less. As a function of LET, the LF probability increases above 10 keV/micrometer, reaching about 5% at around 50 keV/micrometer.

Aerospace Medicine↗

Two-flash photorefraction in screening of amblyogenic refractive errors.

A selection of amblyopic (n = 14) and nonamblyopic (n = 18) children with different ametropias, anisometropias, and astigmatisms was photorefracted without cycloplegia by two-flash photorefraction (T-FP) in which refractions in both horizontal and vertical meridians of both eyes are measured simultaneously. The results were compared to conventional cycloplegic retinoscopy in a blind study. Significant amblyogenic conditions, such as hyperopic anisometropias of more than 1.0 diopter (D), high ametropias, or astigmatism and strabismus cases of 2 degrees to 3 degrees or more are detectable. The technique provides a simple and useful tool for mass screening of infants. The sensitivity of the method for detecting amblyopia seems to be at least as high as in visual checking. Media opacities are recordable, and a permanent document is obtained.

Amblyopia↗

Modulation of flash stimulation intensity and frequency: effects on visual evoked potentials and oscillatory potentials recorded in awake, freely moving mice.

Visual evoked potentials (VEP) responses to flash stimulation at nine intensities, from 0.611 to 945.6 cd/m(2)*s, and two frequencies (0.2 and 1 Hz) were recorded and oscillatory potentials (OPs) extracted after digital 50-Hz high pass filtering in unanaesthetized unrestrained mice. Both VEP and OPs morphology were replicable for all conditions and were similar to values reported in the literature. In particular OPs spectral analysis showed that the main frequency component remained stable at 66-77 Hz, for both stimulation frequencies, although it displayed an increase in amplitude, as a function of stimulus intensity. OPs amplitude at 1 Hz versus 0.2 Hz stimulus frequency was higher after taking into account the different noise contributions in the two conditions. Root mean square values calculated at selected time windows, revealed that, at 1 Hz, the main contribution to OPs occurs at the onset of the response (14-27 ms) while, at 0.2 Hz, the higher RMS was recorded later (42-56 ms). This difference accounts for the longer duration of the oscillatory event in the 0.2-Hz condition and suggests that oscillatory activity, modulated and carried along the visual pathway, is recorded at the cortical electrode after further elaboration at the cortical/subcortical level, depending on stimulus properties.

Animals↗

Flash detection/identification of pathogens, bacterial spores and bioterrorism agent biomarkers from clinical and environmental matrices.

We propose to develop an integrated rapid, semiportable, prototype point microbial detection/identification system for clinical specimens that is also capable of differentiating microbial bioterrorism attacks from threats or hoaxes by defining the pathogen. The system utilizes "flash" extraction/analytical system capable of detection/identification of microbes from environmental and clinical matrices. The system couples demonstrated technologies to provide quantitative analysis of lipid biomarkers of microbes including spores in a system with near-single cell (amol/microl) sensitivity. Tandem mass spectrometry increases specificity by providing the molecular structure of neutral lipids, phospholipids, and derivatized spore-specific bacterial biomarker, 2,6-dipicolinic acid (DPA) as well as the lipopolysaccharide-amide-linked hydroxy-fatty acids (LPS-ALHFA) of Gram-negative bacteria. The extraction should take about an hour for each sample but multiple samples can be processed simultaneously.

Bacteria↗