Evaluation of the Rotating-Frame Relaxation (T1ρ) Filter and Its Application in Metabolomics as an Alternative to the Transverse Relaxation (T2) Filter.

Nuclear magnetic resonance (NMR)-based metabolomic studies commonly involve the use of T2 filter pulse sequences to eliminate or attenuate the broad signals from large molecules and improve spectral resolution. In this paper, we demonstrate that the T1ρ filter-based pulse sequence represents an interesting alternative because it allows the stability and the reproducibility needed for statistical analysis. The integrity of the samples and the stability of the instruments were assessed for different filter durations and amplitudes. We showed that the T1ρ filter pulse sequence did not induce sample overheating for a filter duration of up to 500 ms. The reproducibility was evaluated and compared with the T2 filter in serum and liver samples. The implementation is relatively simple and provides the same statistical and analytical results as those obtained with the standard filters. Regarding tissues analysis, because the duration of the filter is the same as that of the spin-lock, the synchronization of the echo delays with the magic angle spinning (MAS) rate is no longer necessary as for T2 filter-based sequences. The results presented in this article aim at establishing a new protocol to improve metabolomic studies and pave the way for future developments on T1ρ alternative filters, in liquid and HR-MAS NMR experiments.

Conformation changes of cuttlefish (Euprymna morsei) rhodopsin following photoconversion.

Cuttlefish (Euprymna morsei) rhodopsin solubilized in lauryl ester of sucrose and its photoproduct, acid metarhodopsin, were examined by small-angle X-ray scattering and chromatofocusing to investigate the conformation changes of visual pigment following photoconversion. From spectroscopic studies, it was found that more than 93% of Euprymna rhodopsin could be converted to meta form under the condition of red light irradiation at neutral pH. Since almost pure acid metarhodopsin solution was prepared without changing the specimen concentration, the small-angle X-ray scattering intensities of both pigment-detergent complexes were directly compared. The radius of gyration increased on going from rhodopsin to acid metarhodopsin by approximately 1.5%. There were also discernible changes in the secondary peak intensities. The distribution function, derived by the Fourier transformation of intensity data, showed a significant change around 55 A. The maximum linear dimension of the rhodopsin-detergent complex was about 95 A and hardly changed after illumination. Intensity at zero angle did not change after illumination, suggesting that the aggregation did not occur. The change of the intensity profile could be due to the conformational change of the pigment-detergent monomers. The pI value of rhodopsin determined by chromatofocusing was 5.32 and that of acid metarhodopsin was 5.06, indicating that a few carboxyl groups are newly dissociated. The shift of the protein mass and the charge redistribution were observed following photoconversion.

Amino acid sequence of the retinal binding site of squid visual pigment.

The retinylpeptides of visual pigments of two species of squid were identified in invertebrate visual pigments. Their primary structures were identical: H-Phe-Ala-Lys-Ala-Ser-Ala-Ile-His-Asn-pro-Hse(Met)-OH. The sequence was homologous to those of the corresponding region of other visual pigments, but the eighth amino acid, His, was found in squid visual pigments. In this experiment the retinylpeptides of eleven amino acid residues were isolated by monitoring the absorbance spectrum of the reduced retinal Schiff base without using radio-active [3H]retinal. This method is valid for the isolation and identification of retinylpeptides of other invertebrate visual pigments in which the chromophore is not exchangeable.

4-Hydroxyretinal, a new visual pigment chromophore found in the bioluminescent squid, Watasenia scintillans.

The bioluminescent squid, Watasenia scintillans has three visual pigments. The major pigment, based on retinal (lambda max 484 nm), is distributed over the whole retina. Another pigment based on 3-dehydroretinal (lambda max approximately 500 nm) and the third pigment (lambda max approximately 470 nm) are localized in the specific area of the ventral retina just receiving the downwelling light. Visual pigment was extracted and purified from the dissected retina. The chromophores were then extracted and analyzed with HPLC, NMR, infrared and mass spectroscopy, being compared with the synthetic 4-hydroxyretinal. A new retinal derivative, 11-cis-4-hydroxyretinal, is identified as the chromophore of the third visual pigment of the squid.

Adaptation of a deep-sea cephalopod to the photic environment. Evidence for three visual pigments.

Watasenia scintillans, a bioluminescent deep-sea squid, has a specially developed eye with a large open pupil and three visual pigments. Photoreceptor cells (outer segment: 476 micron; inner segment: 99 micron) were long in the small area of the ventral retina receiving downwelling light, whereas they were short (outer segment: 207 micron; inner segment: 44 micron) in the other regions of the retina. The short photoreceptor cells contained the visual pigment with retinal (lambda max approximately 484 nm), probably for the purpose of adapting to their environmental light. The outer segment of the long photoreceptor cells consisted of two strata, a pinkish proximal area and a yellow distal area. The visual pigment with 3-dehydroretinal (lambda max approximately 500 nm) was located in the pinkish proximal area, giving high sensitivity at longer wavelengths. A newly found pigment (lambda max approximately 471 nm) was in the yellow distal area. The small area of the ventral retina containing two visual pigments is thought to have a high and broad spectral sensitivity, which is useful for distinguishing the bioluminescence of squids of the same species in their environmental downwelling light. These findings were obtained by partial bleaching of the extracted pigment from various areas of the retina and by high-performance liquid chromatographic analysis of the chromophore, complemented by microscopic observations.

X-ray diffraction study of the live squid retina.

In previous studies of invertebrate rhabdomes by X-ray diffraction, glutaraldehyde fixation of the retina was used because this tissue is very labile and, without fixation, disintegrates within an hour of dissection. However, with conventional X-ray apparatus more than ten hours exposure time was needed to record a diffraction pattern. In this study, X-ray diffraction patterns from unfixed squid retina could be successfully obtained by use of synchrotron radiation and a storage phosphor screen as detector. The diffraction spots were indexed on a two-dimensional hexagonal lattice of 60 nm. X-ray data was analysed by comparing Patterson functions calculated from the diffraction intensities with those based on model building. The hexagonal shape of microvillar cross section was suggested by the systematic weakness of (0, k) reflections beyond k = 4 and the appearance of the six symmetry-related diffuse maxima around (4 nm)-1. The best-fitting model showed a large gap between adjacent microvilli (approximately 12 nm), which has been expected (for ionic current flow through the inter-microvillus space to generate the membrane potential) but not observed with the chemically fixed retina, possibly due to an artifact of fixation. Also, the existence of massive inter-microvillus material, scarcely observed by conventional electron microscopy, has been confirmed.

STRUCTURAL BASIS FOR WAVELENGTH DISCRIMINATION IN THE BANKED RETINA OF THE FIREFLY SQUID WATASENIA SCINTILLANS

There is a greatly thickened region of retina in the ventral part of the eye of the firefly squid Watasenia scintillans, with an outer segment (OS) layer around 600 µm thick. The distal two-thirds of this OS layer is yellow and contains a visual pigment, based on 4-hydroxyretinal (A4), with an absorbance maximum at 470 nm. The proximal third is pink and contains a visual pigment, based on dehydroretinal (A2), with an absorbance maximum at 500 nm. Light and electron microscopic investigations demonstrate the presence of four types of photoreceptor cell. In the pink layer, three of the four types (alpha, ß and gamma) produce no microvilli and are columnar in structure. These cells form square microvillous rhabdoms only in the distal yellow layer. The fourth cell type (delta) produces microvilli in the pink layer only. These observations led us to propose that the A2-based visual pigment is contained in the pink-layer cells and that the A4-based visual pigment is contained in the three types of yellow-layer cells. The absorbance of fresh retina was determined by microspectrophotometry. The yellow OS layer, with an absorbance of 0.7 per 100 µm thickness at 470 nm, is expected to act as a short-wavelength cut-off filter to the underlying pink OS cells, shifting their photosensitivity peak by an estimated 50 nm to 550 nm. Possible wavelength discrimination by this squid is discussed.

Light-induced, GTP-binding protein mediated membrane currents of Xenopus oocytes injected with rhodopsin of cephalopods.

Xenopus oocytes that were injected with rhabdomeric membranes of squid and octopus photoreceptors acquired light sensitivity. The injected oocytes showed a light-induced current having characteristics similar to other G-protein-mediated Cl- currents induced by the activation of other membrane receptors. Pretreatment of the oocytes with pertussis toxin before the injection suppressed the generation of the light-induced current, indicating an ability of cephalopod rhodopsin to cross-react with an endogenous G-protein of Xenopus oocytes.

The absorbance spectrum and photosensitivity of a new synthetic "visual pigment" based on 4-hydroxyretinal.

The firefly squid, Watasenia scintillans, is the only animal known to possess a visual pigment in which the chromophore is 4-hydroxyretinal. This paper describes the absorbance spectrum and some properties of a synthetic "A4" visual pigment generated from bovine opsin and 4-hydroxyretinal. The absorbance spectrum of this pigment is compared with (a) bovine rhodopsin and (b) a rhodopsin template with the same lambda max as the synthetic visual pigment. The A4 pigment is shown to have an absorbance spectrum that is almost identical to that of a rhodopsin template. It is also shown that the photosensitivity and thermal stability of the A4 pigment, dispersed in detergent micelles, is essentially similar to that of rhodopsin.

Structural and functional differences of two forms of GTP-binding protein, Gq, in the cephalopod retina.

The major GTP-binding protein (G-protein) in the rhabdomeric photoreceptor membranes of the squid (Watasenia scintillans) has been identified as a Gq-class G-protein. Anti-Gq alpha antibodies recognized a protein not only in the photoreceptor membranes but also in soluble fractions of the retina. The 42 kD protein in the soluble fractions (soluble Gq alpha) had the same molecular mass and the same reactivities to anti-Gq antibodies as those of membrane-bound Gq alpha. The G beta subunit was scarcely detected in the soluble fractions, being found mostly in the membrane fraction, indicating soluble Gq alpha exists in monomeric form. Soluble Gq alpha had no effect on the GTPase activity of the photoreceptor membranes, suggesting that it does not interact with photoactivated rhodopsin or G beta gamma. Soluble Gq alpha would be an inactive form of Gq alpha. In the retina of Octopus fangsiao, soluble Gq alpha was scarcely detected after dark adaptation, but increased during subsequent light exposure and decreased on returning to dark adaptation. These results with Octopus suggest that functional membrane-bound Gq alpha is converted to soluble Gq alpha on exposure to light. Transformation of membrane-bound Gq alpha into the soluble form by hydroxylamine suggests that the difference between membrane-bound and soluble Gq alpha is associated with the attachment of fatty acid(s).