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At least 757 records · Page 42Linked to original sources
PubMed2005

A structural pathway for signaling in the E46Q mutant of photoactive yellow protein.

In the bacterial photoreceptor photoactive yellow protein (PYP), absorption of blue light by its chromophore leads to a conformational change in the protein associated with differential signaling activity, as it executes a reversible photocycle. Time-resolved Laue crystallography allows structural snapshots (as short as 150 ps) of high crystallographic resolution (approximately 1.6 A) to be taken of a protein as it functions. Here, we analyze by singular value decomposition a comprehensive time-resolved crystallographic data set of the E46Q mutant of PYP throughout the photocycle spanning 10 ns-100 ms. We identify and refine the structures of five distinct intermediates and provide a plausible chemical kinetic mechanism for their inter conversion. A clear structural progression is visible in these intermediates, in which a signal generated at the chromophore propagates through a distinct structural pathway of conserved residues and results in structural changes near the N terminus, over 20 A distant from the chromophore.

Bacterial Proteins↗
PubMed2004

On the relation between the photoactivation energy and the absorbance spectrum of visual pigments.

We relate the collected experimental data on the minimum energy for photoactivation (E(a)) to the wavelengths of peak absorbance (lambda(max)) of 12 visual pigments. The E(a) values have been determined from the temperature-dependence of spectral sensitivity in the long-wavelength range. As shown previously, the simple physical idea E(a) =const. x (1/lambda(max)) (here termed the Stiles-Lewis-Barlow or SLB relation) does not hold strictly. Yet there is a significant correlation between E(a) and 1/lambda(max) (r(2)=0.73) and the regression slope obtained by an unbiased fit is 84% of the predicted value of the best SLB fit. The correlation can be decomposed into effects of A1 --> A2 chromophore change and effects of opsin differences. For a chromophore change in the same opsin, studied in two A1/A2 pigment pairs, the SLB relation holds nearly perfectly. In seven pigments having different opsins but the same (A2) chromophore, the correlation of E(a) and 1/lambda(max) remained highly significant (r(2)=0.78), but the regression coefficient is only 72% of the best SLB fit. We conclude that (1) when the chromophore is exchanged in the same opsin, the lambda(max) shift directly reflects the difference in photoactivation energies, (2) when the opsin is modified by amino acid substitutions, lambda(max) and E(a) can be tuned partly independently, although there is a dominant tendency for inverse proportionality. In four (A1) rhodopsins with virtually the same lambda(max), E(a) varied over a 4.5 kcal/mol range, which may be taken as a measure of the freedom for independent tuning. Assuming that low E(a) correlates with high thermal noise, we suggest that the leeway in lambda(max) - E(a) coupling is used by natural selection to keep E(a) as high as possible in long-wavelength-sensitive pigments, and that this is why the opsin-dependent E(a) (1/lambda(max))-relation is shallower than predicted.

Animals↗
PubMed2006

Late stages of visual pigment photolysis in situ: cones vs. rods.

Slow photolysis reactions and the regeneration of the dark pigment constitute the mechanisms of dark adaptation whereby photoreceptor cells restore their sensitivity after bright illumination. We present data on the kinetics of the late stages of the photolysis of the visual pigment in intact rods and red- and green-sensitive cones of the goldfish retina. Measurements were made on single photoreceptors by means of a fast-scanning dichroic microspectrophotometer. We show that in cones the hydrolysis of the opsin-all-trans 3-dehydroretinal linkage proceeds with a half-time of approximately 5s at 20 degrees C that is almost two orders of magnitude faster than in rods. 3-Dehydroretinol in cones is produced approximately 3-fold faster than retinol in amphibian rhodopsin rods; the rate of the reaction is limited by the speed of retinal reduction catalyzed by retinoldehydrogenase. The fast hydrolysis of the 3-dehydroretinal/opsin Schiff base and the correspondingly fast appearance of the substrates for dark visual pigment regeneration (free opsin and 3-dehydroretinol) provide essential conditions for faster dark adaptation of cone (diurnal) as compared to rod (nocturnal) vision.

Animals↗
PubMed1997

A microspectrophotometric study of the shielding properties of eyespot and cell body in Chlamydomonas.

The eyespot apparatus of the unicellular alga Chlamydomonas exhibits a clear directivity, i.e., it perceives light from different directions with different sensitivity. Using a newly constructed confocal microscope we have studied how absorption and reflection of eyespot and cell body shape this directivity. In agreement with previous results the eyespot was found to be highly reflectant, owing to its interference reflector design, but only for yellow light. Light of 490 nm, the maximum of absorption of the photoreceptor, was hardly reflected at all, even when the reflector was "tuned" to lower wavelengths by tilting it relative to the incoming light. The absorption of the carotenoids in the interference reflector also contributed little to the shielding properties of the cell, leaving the major contribution to the cell body. Thus most of the attenuation of light reaching the eyespot from the rear is due to chlorophyll and other pigments within the cell. In its peak around 490 nm the "contrast-ratio" reached a value of 8-10.

Animals↗
PubMed1995

Rod outer segment (ROS) renewal as a mechanism for adaptation to a new intensity environment. II. Rhodopsin synthesis and packing density.

Using intraocular injection of a 3H-phe and 3H-leu mixture, we found that the net synthesis of rhodopsin is light-intensity-dependent and can adjust when an animal encounters a new lighting environment. Rhodopsin net synthesis dropped dramatically in day 1, 200-lx immigrants; however, preliminary studies show that the opsin mRNA levels in these animal were the same as that found in the 200-lx-native group. This suggests that the changes in the net synthesis of rhodopsin found when an animal is moved to a higher intensity light may be controlled at some point post-transcriptionally. Microspectrophotometric measurements on single rod cells revealed that the differences in whole-eye rhodopsin levels between the two cyclic intensity groups, 3 lx and 200 lx, were also present at the single cell level. This supports previous studies suggesting that the density of rhodopsin per disc varies according to the intensity of light to which the animal is exposed. Individual rods also had differences in packing density of rhodopsin at the base compared to the tip of the same outer segment when the animal had been switched to a new intensity for one half of a turnover period (5 days). This indicates the amount of rhodopsin per disc is regulated and suggests that renewal is the process responsible for creating a modified outer segment. Animals, when switched to a new intensity, can adjust the synthesis of rhodopsin and the number of molecules added per disk to ensure the appropriate packing density of rhodopsin in the rod outer segment (ROS) disk membranes for that level of light intensity.

Adaptation, Ocular↗
PubMed1998

X-ray and spectrophotometric studies of the binding of proflavin to the S1 specificity pocket of human alpha-thrombin.

Proflavin can be used to study the interactions of inhibitors and substrates with thrombin by monitoring the changes in the visible absorption spectrum that occur on dye displacement. We have used microspectrophotometric methods to investigate the binding of proflavin to crystals of an alpha-thrombin-hirugen complex and have determined the structure by X-ray crystallography. The proflavin molecule binds in the S1 pocket of the enzyme with one of the amino groups hydrogen bonded to the carboxylate of Asp-189 while the protonated ring nitrogen is hydrogen bonded to the carbonyl of Gly-219. This result indicates that the proflavin displacement assay can be used to specifically monitor the binding of inhibitors to the S1 pocket.

Absorption↗
PubMed2001

Ionochromic properties of long-wave-sensitive cones in the goldfish retina: an electrophysiological and microspectrophotometric study.

Long-wave-sensitive (LWS) cone visual pigments are sensitive to the concentration of chloride ions and show a spectral shift to shorter wavelengths when exposed to low chloride levels. We have used the aspartate-isolated mass receptor potential of the electroretinogram (ERG) to establish whether the spectrally shifted cone pigment is functional. In the goldfish, Carassius auratus, the lambda(max) of the LWS porphyropsin is displaced from about 622 nm to around 606 nm when chloride is replaced by gluconate. The electrical response of the LWS cones (but not MWS cones and rods) is selectively and reversibly abolished by the removal of chloride ions. The effect is concentration dependent and a decrease to half the normal chloride ion concentration is sufficient to cause a decrease in the response.

Animals↗
PubMed1999

An ultraviolet absorbing pigment causes a narrow-band violet receptor and a single-peaked green receptor in the eye of the butterfly Papilio.

The distal photoreceptors in the tiered retina of Papilio exhibit different spectral sensitivities. There are at least two types of short-wavelength sensitive receptors: an ultraviolet receptor with a normal spectral shape and a violet receptor with a very narrow spectral bandwidth. Furthermore, a blue receptor, a double-peaked green receptor and a single-peaked green receptor exist. The violet receptor and single-peaked green receptor are only found in ommatidia that fluoresce under ultraviolet illumination. About 28% of the ommatidia in the ventral half of the retina exhibit the UV-induced fluorescence. The fluorescence originates from an ultraviolet-absorbing pigment, located in the most distal 70 microns of the ommatidium, that acts as an absorption filter, both for a UV visual pigment, causing the narrow spectral sensitivity of the violet receptor, and for a green visual pigment, causing a single-peaked green receptor.

Animals↗
PubMed1999

Morphological changes in the retina of Aequidens pulcher (Cichlidae) after rearing in monochromatic light.

We investigate the processing of chromatic information in the outer retina of a cichlid fish, Aequidens pulcher. The colour opponent response characteristics of some classes of cone-specific horizontal cells in the fish retina are the result of feedforward-feedback loops with cone photoreceptors. To interfere with the reciprocal transmissions of signals, animals were reared in monochromatic lights which preferentially stimulated the spectrally different cone types. Here we report the effects on the cones. Their absorbance spectra were largely unaffected, indicating no change in photopigment gene expression. Significant changes were observed in the cone outer segment lengths and the frequencies of spectral cone types. Quantum catch efficiency and survival of cones appear to be controlled in a spectrally selective way. Our results suggest that the retina responds to spectral deprivation in a compensatory fashion aimed at balancing the input from the different cone types to second order neurons.

Animals↗
PubMed2000

Spectral sensitivity of photoreceptors in an Australian marsupial, the tammar wallaby (Macropus eugenii).

Microspectrophotometric measurements on the rod photoreceptors of the tammar wallaby showed that they have a peak absorbance at 501 nm. This indicates that macropod marsupials have a typical mammalian rhodopsin. An electroretinogram-based study of the photoreceptors confirmed this measurement and provided clear evidence for a single middle wavelength-sensitive cone pigment with a peak sensitivity at 539 nm. The electroretinogram did not reveal the presence of a short-wavelength-sensitive cone pigment as was expected from behavioural and anatomical data. Limitations of the electroretinogram in demonstrating the presence of photopigments are discussed in relation to similarly inconsistent results from other species.

Animals↗
PubMed2001

Heterogeneous distribution of single-bottle adhesive monomers in the resin-dentin interdiffusion zone.

OBJECTIVES: The purpose of this study was to investigate whether monomer separation occurs in single-bottle adhesives applied on acid-etched dentin surfaces. METHODS: Smear-layer covered dentin specimens produced from sound premolars, were acid-etched and analyzed by micro-MIR FTIR spectroscopy, before and after treatment with the single-bottle adhesives One-Step, Prime and Bond 2.1, Scotchbond 1 and Syntac-Sprint. The difference spectra (etched, and primed minus etched of the same dentin surfaces) were plotted and compared with original adhesive spectra. The C=C/C...C (aromatic adhesives) and C=C/C=O (aliphatic adhesives) peak area ratios were used to evaluate the extent of monomer separation relative to original adhesive spectra at the uppermost 2 microm of the resin-dentin interdiffusion zone. Three dentin specimens were used for each adhesive. One-way ANOVA and Newman-Keuls tests were performed to assess the statistically significant differences (alpha=0.05). RESULTS: All the adhesives demonstrated separation of monomer components on etched dentin ranging in mean values from 68.7 to 81.9% relative to reference. High molecular weight hydrophobic (i.e. BisDMA, BisGMA, BisGMA adducts) and hydrophilic monomers (i.e. BPDMA) predominated at the region probed. Prime and Bond 2.1 demonstrated the highest extent of monomer separation. SIGNIFICANCE: Heterogeneous monomer distribution following application to etched dentin may affect the curing performance of single-bottle adhesives within the resin infiltrated region and consequently, the mechanical and chemical stability of the network formed.

Acid Etching, Dental↗
PubMed2000

The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays.

Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center. Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form. Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum. Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.

Crystallization↗
PubMed2003

FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus.

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between normal primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because primary cells are similar to normal tissue cells in most of their characteristics. Our results showed dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis of the FTIR-MSP spectra, also displayed significant differences between the control and transformed cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra yielded 100% accuracy in classifying both types of cells.

Amides↗
PubMed2004

A high performance micro-dual-wavelength-spectrophotometer (MDWS).

The dual wavelength spectrophotometer (DWS) has proven to be the most sensitive device to monitor minute optical absorbance changes, which are inaccessible to conventional single or double beam spectrophotometers. The typical set ups, e.g. extensively used for Ca2+ or phytochrome measurements, are huge, expensive and cumbrous. Therefore, a novel high performance micro-dual-wavelength spectrophotometer (MDWS) was developed. It is miniaturized and no moving parts such as vibrational mirrors or rotating filter wheels involved. Its specifications are superior compared to the conventional set up being capable of detecting minute optical changes (reflection, absorbance, transmittance) at particular wavelengths.

Calibration↗
PubMed1998

A fluorescence-based method for assessing dural protein extravasation induced by trigeminal ganglion stimulation.

Neurogenic dural inflammation has been proposed as a source of pain during migraine. Unilateral electrical stimulation of the trigeminal ganglion causes the ipsilateral release of inflammatory neuropeptides and subsequent dural plasma protein extravasation, a component of neurogenic inflammation. We measured the amount of protein leaking into the dural tissue of guinea pigs following trigeminal ganglion stimulation by exploiting the complexation reaction of endogenous proteins with the fluorescent dye Evans Blue, instead of utilizing exogenous radiolabeled albumin as commonly done in the literature. The amount of Evans Blue trapped in dural tissue following electrical stimulation of the trigeminal ganglion was measured using a fluorescence microscope equipped with a spectrophotometer. This method utilized multiple measurements on each dura sample which resulted in very precise values using a small number of animals per point (n = 3). Sumatriptan and CP-122,288 were found to dose-dependently prevent neurogenic dural extravasation. The potencies of CP-122,288 and sumatriptan were found to be similar to those reported in the literature when similar experimental protocols were used.

Animals↗
PubMed1998

A microspectrophotometric study on the physicochemical properties of antipyrylazo III injected into rat myoballs for measuring free magnesium ion.

The accurate measurement of the intracellular concentration of free magnesium ions ([Mg2+]i) is essential for evaluating the role of Mg2+ in cellular functions. Among the specific (compared to fluorescent indicators) metallochromic dyes, antipyrylazo III (APIII) appears to be most suitable for measuring such concentrations in vertebrate cells according to in vitro studies. In this work, the intracellular physicochemical properties of APIII as a Mg2+ indicator were investigated in the cultured rat myoball by means of a microspectrophotometric technique, in order to obtain an accurate measurement of [Mg2+]i. A set of intracellular APIII-Mg2+ calibration spectra was established after permeabilization of the cell membrane with saponin. In comparison with recordings obtained in K+ solutions, the APIII absorption spectra recorded on a myoball exhibited a red shift and an overall change in absorbance, similar to that observed in a protein (bovine serum albumin: BSA) solution. The apparent dissociation constant of APIII for Mg2+ in the myoplasm was found to be 3.16 mM, significantly higher than the 1.86 mM measured in K+ solutions at the same pH (7.35). A stoichiometric ratio of 1:1 was found, however, both in solution and in the myoplasm. In addition, the injected APIII significantly affected the [Mg2+]i of myoballs. The [Mg2+]i was found to be 0.9+/-0.18 mM in 85 myoballs, on the basis of the intracellular calibration spectra obtained at the same myoplasmic APIII concentration ( approximately 2.5 mM), and after correction for the perturbing effect of the dye. It is concluded that an intracellular calibration and recording of whole spectrum are essential for proper interpretations of intracellular dye signals.

Animals↗
PubMed2001

Microspectrofluorometry of autofluorescence emission from human leukemic living cells under oxidative stress.

Image cytometry was applied to study the intracellular localization of autofluorescence and the influence of an oxidative stress on this emission. K562 erythroleukemia cancer cells were analyzed with a microspectrofluorometer, coupled with a Argon laser (Ar+) (363 nm). From each cell, 15 x 15 emission spectra were recorded in the 400-600 nm spectral range to generate a spectral image of autofluorescence. The intracellular locations of the autofluorescence emission and of the specific mitochondrial probe rhodamine 123 (R123) were matched. Under a 363 nm excitation, all spectra from K562 cells show equivalent profiles with a 455 nm maximum emission, near of reduced nicotinamide adenine dinucleotide-(Phosphate) solution (NAD(P)H) (465 nm maximum emission). The spatial distribution of autofluorescence is homogeneous and different from the one of R123. Hydrogen peroxide (H2O2) (200 microM) and menadione (Men) (5 microM) induce a weak spectral change and a decrease in autofluorescence intensity, down to 40% of the initial emission. Doxorubicin (Dox) induces a dose-dependent decrease in autofluorescence emission and a release of intracellular free radicals. When cells were pre-treated 1 h with 1 mM glutathione (GSH), Dox induces a lower free radicals release, no significant variation of autofluorescence intensity and a lower growth inhibitory effect. Images cytometry of autofluorescence suggest that the intracellular NAD(P)H would not be restricted to mitochondrial compartments. The release of free radicals was associated with a decrease in autofluorescence intensity, mainly attributed to NAD(P)H oxidation both inside and outside mitochondria.

Dose-Response Relationship, Drug↗