Origin of the vertebrate visual cycle

In vertebrates, the absorption of light by rhodopsin leads to the isomerization of 11-cis-retinal chromophore to its all-trans form. In the visual cycle, all-trans retinal is converted back to 11-cis retinal. Mammalian visual cycle takes place in photoreceptor cells and retinal pigment epithelial (RPE) cells, while that of cephalopods is completed within a photoreceptor cell. To identify visual cycle system in the primitive chordate ascidians, we studied the localization of the ascidian visual cycle genes and proteins by in situ hybridization and whole-mount immunohistochemistry, respectively. We identified four genes encoding putative visual cycle proteins, homologs of retinal G protein-coupled receptor (Ci-opsin3), cellular retinaldehyde-binding protein (Ci-CRALBP), beta-carotene 15,15'monooxygenase (Ci-BCO) and RPE-specific 65 kDa protein (Ci-RPE65) in the ascidian, Ciona intestinalis. In contrast to Ci-BCO, which is predominantly localized in ocellus photoreceptor cells of the larva, Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable with that of Ci-opsin3 and Ci-CRALBP. Proteins of Ci-opsin3, Ci-CRALBP and Ci-BCO are localized in photoreceptor cells. These results suggest that the larval visual cycle uses Ci-opsin3 as a photoisomerase, while the visual cycle of the adult photoreceptors is RPE65-dependent. The colocalization of visual cycle proteins in the photoreceptor cells suggest that ascidian visual cycle takes place in a photoreceptor cell as seen in the cephalopod visual cycle.     

RPE65 and the Accumulation of Retinyl Esters in Mouse Retinal Pigment Epithelium

The RPE65 protein of the retinal pigment epithelium (RPE) enables the conversion of retinyl esters to the visual pigment chromophore 11‐cis retinal. Fresh 11‐cis retinal is generated from retinyl esters following photoisomerization of the visual pigment chromophore to all‐trans during light detection. Large amounts of esters accumulate in Rpe65^?/? mice, indicating their continuous formation when 11‐cis retinal generation is blocked. We hypothesized that absence of light, by limiting the conversion of esters to 11‐cis retinal, would also result in the build‐up of retinyl esters in the RPE of wild‐type mice. We used HPLC to quantify ester levels in organic extracts of the RPE from wild‐type and Rpe65^?/? mice. Retinyl ester levels in Sv/129 wild‐type mice that were dark adapted for various intervals over a 4‐week period were similar to those in mice raised in cyclic light. In C57BL/6 mice however, which contain less Rpe65 protein, dark adaptation was accompanied by an increase in ester levels compared to cyclic light controls. Retinyl ester levels were much higher in Rpe65^?/? mice compared to wild type and kept increasing with age. The results suggest that the RPE65 role in retinyl ester homeostasis extends beyond enabling the formation of 11‐cis retinal.     

Identification and characterization of homologues of vertebrate beta-thymosin in the marine mollusk Aplysia californica

The beta-thymosins have been known as actin-sequestering proteins, but now are recognized as molecules with multiple and diverse intracellular and extracellular functions. Two closely related proteins, beta-thymosin(His) and beta-thymosin(Gln), have been de novo sequenced by top-down mass spectrometry in the common neurobiology model, Aplysia californica. As determined by nanoelectrospray quadrupole-enhanced Fourier-Transform mass spectrometry with collisionally activated and electron-capture dissociations, both of these Aplysia beta-thymosins are acetylated and differ by a single residue in the central actin-binding domain. Profiling of individual cells and tissue by matrix-assisted laser desorption/ionization mass spectrometry reveals that these proteins are widely expressed in the Aplysia central nervous system, including in individual identified neurons, neuronal clusters, nerves and connective tissues. Newly identified beta-thymosin(His) and beta-thymosin(Gln) are also detected by mass spectrometry in hemolymph, and in releasates collected from whole ganglia. When applied exogenously, beta-thymosin proteins, purified from nerve cell extract, support the anchoring of neurons, and increase neurite sprouting and total neurite outgrowth in culture. These positive effects on neurite regeneration in cell culture suggest that the beta-thymosin proteins have an extracellular function in the central nervous system of Aplysia californica.     

Controlling factors in the synthesis of cucurbituril and its homologues.

The acid-catalyzed synthesis of cucurbit[n]urils from formaldehyde and glycoluril is poorly understood. In this paper, we examine a wide range of reaction conditions that include the effects of acid type, acid concentration, reactant concentrations, and temperature to both probe the mechanism and optimize the yields of isolated cucurbit[n]urils, where n = 5-10. A mechanism for the formation of these cucurbit[n]urils is presented. Individual cucurbit[n]urils were unambiguously identified in reaction mixtures using ESMS and (13)C NMR.     

Organometallic complexes with biological molecules. XV. Effects of tributyltin(IV)chloride on enzyme activity,Ca2+, and biomolecule and synthesis in Ciona intestinalis (Urochordata) ovary

Considerable attention has been given in recent years to the possibility that xenobiotics in the environment may affect reproduction in animals. In this study, the relative impact of tributyltin(IV) (TBT) chloride, one of the most toxic environmental pollutants, was investigated using Ciona intestinalis ovary as a model system. The pleiotropic effects of TBT exposure are concentration dependent and include a decrease of ATP levels, lipid content and nucleic acid content and synthesis. In contrast, a marked increase in calcium (Ca²⁺) and glucose content is observed. Furthermore, TBT alters enzymatic activity, inhibiting creatine kinase and stimulating alkaline phosphatase and cholinesterase (at concentrations higher than 10^?5M in sterile sea water solution). The implications of these effects on reproduction and embryonal development are discussed, along with the possibility that they reflect an extreme cellular defence mechanism triggered to avoid deleterious consequences for the survival of the species.Copyright © 2001 John Wiley & Sons, Ltd.     

Determination of the distribution of cannabinoids in cannabis resin from the Lebanon using HPLC. Part III

Samples taken from 61 slabs of cannabis resin originating in The Lebanon were examined by high performance liquid chromatography. The slabs were derived from seizures of cannabis resin made over a three-year period. From a study of 14 of these samples, taken from bags which bore a number of identical stamp marks, it was shown that there was a correlation between the cannabinoid distribution and the marks. The remaining 47 samples of cannabis resin, all of which were from bags with different marks, were each found to have a unique cannabinoid distribution. Such an examination can therefore be used to aid investigations where evidence of a common origin is sought.     

Vibrational—rotational structure in the angular distribution and intensity of photoelectrons from diatomic molecules. III. Effects of higher partial

Total and differential cross sections for the photoionization of H₂ are calculated for each vibrational—rotational transition induced in the molecule. The transition moment is evaluated in the two-center spheroidal coordinates with varying internuclear distance. The wavefunction for the ejected electron is calculated with partial-wave coupling taken into account. It is found that the calculation with only the lowest partial wave retained gives a sufficiently reliable result for most of the cross sections for 584 A and 736 A photons. Only the exception is the anisotropy parameter for the photoelectron angular distribution in the case where rational transition |ΔJ| = 2 occurs. The parameter, especially for fast photoelectrons, increases very much when higher partial waves are included in the calculation. Some preliminary results for the incidence of 304 A line are also shown.     

Interdigitated smectic A and B mesophases in higher homologues of the 5O.m series

We report the synthesis, characterization and structural studies of the higher homologues of N(4-n-pentyloxybenzylidene)4-n-alkylaniline compounds, viz. 5O.12, 5O.14 and 5O.16 by thermal microscopy, differential scanning calorimetry and X-ray diffraction. The compounds exhibit N-S_A-S_B, N-S_A-S_B and N-S_A phase sequence variants, respectively. The X-ray results reveal the existence of an interdigitated smectic A phase in 5O.16 and smectic B phase in 5O.14. Results are discussed in light of the available data on the nO.m series     

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. III: pH 2.5-4 intervals

To study the molecular mass distribution and number of species in narrow-range (2-pH-unit wide, in the nominal pI 2-4 or 3-5 interval) carrier ampholytes from four commercial sources (Bio-Lyte, Servalyt, Ampholine and Pharmalyte), a 2-D technique was adopted, consisting of a preparative focusing step in a Rotofor instrument, followed by analysis of every other collected fraction (10 out of 20) by CE-MS. It was found that Ampholine pH 3.5-5 contains 105 different molecular mass (M(r)) compounds, in the M(r) interval 205-965 Da, for a total of 446 isoforms. Bio-Lyte pH 3-5 consists of 84 different M(r) species, in the M(r) range 216-965 Da, for a total of 383 isoforms. Servalyt pH 2-4 is made of 227 different M(r) compounds, in the M(r) interval 204-929 Da, for a total of 1201 isoforms. Pharmalyte pH 2.5-5 comprises 245 amphoteres, in the M(r) range 203-857 Da, for a total of 857 isoforms. Pharmalyte appears to be the best brand, with the vast majority of species focusing sharply at their pI position and almost no 'poor' species, distributed along the entire pH gradient, denoting an extremely shallow pH/mobility curve across the pI value. Due to some overlap with the adjacent acidic pH 4-6 interval, the species in common have been evaluated: the most extended overlaps are found in Ampholine (55% of the species appearing in the two neighbouring intervals) and in Servalyt (47% coincidence). The lowest overlaps are found in Pharmalyte (23%) and in Bio-Lyte (20%).     

Electron distribution in iron octaethyloxophlorin complexes. Importance of the Fe(III) oxophlorin trianion form in the bis-pyridine and bis-imidazole complexes

The apportionment of electrons between iron and the porphyrinic macrocycle in complexes of octaethyloxophlorin (H3OEPO) has been a vexing problem. In particular, for (Py)2Fe(OEPO), which is an important intermediate in heme degradation, three resonance structures involving Fe(III), Fe(II), or Fe(I), respectively, have been considered. To clarify this matter, the electronic and geometric structures of (Py)2Fe(III)(OEPO), (Im)2Fe(III)(OEPO).2THF, and (Im)2Fe(III)(OEPO).1.6CHCl3 have been examined by single-crystal X-ray diffraction, measurement of magnetic moments as a function of temperature, and EPR and NMR spectral studies. The results clearly show that both complexes exist in the Fe(III)/oxophlorin trianion form rather than the Fe(II)/oxophlorin radical form previously established for (2,6-xylylNC)(2)Fe(II)(OEPO.). In the solid state from 10 to 300 K, (Py)2Fe(III)(OEPO) exists in the high-spin (S = 5/2) state with the axial ligands in parallel planes, a planar porphyrin, and long axial Fe-N distances. However, in solution it exists predominantly in a low-spin (S = 1/2) form. In contrast, the structures of (Im)2Fe(III)(OEPO).2THF and (Im)2Fe(III)(OEPO).1.6CHCl3 consist of porphyrins with a severe ruffled distortion, axial ligands in nearly perpendicular planes, and relatively short axial Fe-N distances. The crystallographic, magnetic, EPR, and NMR results all indicate that (Im)2Fe(III)(OEPO) exists in the low-spin Fe(III) form in both the solid state and in solution.     

Aliphatic polyesters. III. Molecular weight and molecular weight distribution in alcohol-initiated polymerizations of ϵ-caprolactone

The effect of initiation of ?-caprolactone polymerization with mono- and polyfunctional alcohols was investigated. The resulting linear and starshaped polymers were characterized by measurement of the molecular weight and molecular weight distribution. The polymerizations were characterized by (1) rapid initiation, (2) invariance of the number of growing chains corresponding to the amount of initiator, and (3) a dominant role played by ester interchange reactions.     

Size distribution analysis of polymer latex systems by use of the extrema in the angular light scattering pattern. III. Unpolarized and horizontally polarized scattered light

The extrema in the unpolarized and the horizontally polarized angular scattering patterns are used as a basis for size-distribution analysis of polymer latex systems composed of single, spherical, optically isotropic particles. The method of analysis is similar to that recently proposed for vertically polarized scattered light and the polarization ratio, where the modal diameter and a distribution width parameter are determined from the experimental angular scattering pattern and prepared theoretical diagrams obtained by Mie theory calculations. The method of analysis, based on all four scattering functions, is illustrated by use of a Dow polystyrene latex sample.     

Inertial solvent dynamics and the analysis of spectral line shapes: Temperature-dependent absorption spectrum of beta-carotene in nonpolar solvent

The influence of solvent dynamics on optical spectra is often described by a stochastic model which assumes exponential relaxation of the time-correlation function for solvent-induced frequency fluctuations. In contrast, theory and experiment suggest that the initial (subpicosecond) phase of solvent relaxation, resulting from inertial motion of the solvent, is a Gaussian function of time. In this work, we employ numerical and analytical calculations to compare the predicted absorption line shapes and the derived solvent reorganization energies obtained from exponential (Brownian oscillator) versus Gaussian (inertial) solvent dynamics. Both models predict motional narrowing as the ratio kappa = Lambda/Delta is increased, where Lambda and Delta are the frequency and variance, respectively, of the solvent-induced frequency fluctuations. However, the motional narrowing limit is achieved at lower values of kappa for the Brownian oscillator model compared to the inertial model. For a given line shape, the derived value of the solvent reorganization energy lambdasolv is only weakly dependent on the solvent relaxation model employed, though different solvent parameters Lambda and Delta are obtained. The two models are applied to the analysis of the temperature-dependent absorption spectrum of beta-carotene in isopentane and CS2. The derived values of lambdasolv using the Gaussian model are found to be in better agreement with the high temperature limit of Delta2/2kBT than are the values obtained using the Brownian oscillator model. In either approach, the solvent reorganization energy is found to increase slightly with temperature as a result of an increase in the variance Delta of the solvent-induced frequency fluctuations.     

Heterogeneous nucleation in the crystallization of polyolefins. III. Theory and mechansim

The nucleating activity of a large number of nucleating agents for the crystallization of polyolefins is not based on a high surface free energy of the substrate or on epitaxy, or on a minimum particle size just compatible with the thickness of the crystallizing polymer lamellae, or on any persistance of crystalline material in holes of the nucleating particles. The relevant mechanism is thought to be nucleation at steps of limited length in the surface of the nucleating particles. The nucleation is enhanced by a high degree of accommodation of polymer molecules at the surface of the substrate through the presence of ditches in the latter which cause prealignment of polymer chains and thus facilitate crystallization.     

Method for the determination of beta-carotene in supplements and raw materials by reversed-phase liquid chromatography: single laboratory validation

A single laboratory validation (SLV) study was conducted for a liquid chromatography (LC) method for the determination of total and all-trans-beta-carotene in a variety of dietary supplements, including multivitamin tablets, softgels, capsules, and beadlet raw materials. Extraction variants were developed for the different types of supplements tested based upon the supplement type and level of beta-carotene. Water dispersible formulations such as powders, emulsions, tablets, and capsules were enzymatically digested with protease and extracted with dichloromethane-ethanol. Oily suspensions were directly dissolved in dichloromethane-ethanol. After appropriate dilution or concentration, the extracts were chromatographed by using either a reversed-phase C18 column or, in products containing high amounts of alpha-carotene, a reversed-phase C30 column. The LC systems provided linear responses in the range of 0.1-50 microg beta-carotene/mL. The main geometrical isomers of beta-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were well separated from each other and from other carotenoids such as a-carotene, cryptoxanthin, lutein, lycopene, and zeaxanthin. Duplicate determinations of total beta-carotene performed by 2 technicians in 8 different test materials on 5 different days resulted in relative standard deviations of 1.2-4.4%. Recoveries determined for supplements and beadlet raw material spiked with beta-carotene levels of 10 microg to 100 mg/test portion and 0.2-40%, respectively, ranged from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results from the SLV study, the method is suggested for a collaborative study on the determination of total and all-trans-beta-carotene in dietary supplements.