Embryonic nutritional hyperglycemia decreases cell proliferation in the zebrafish retina.

Diabetic retinopathy (DR) is one of the leading causes of blindness in the world. While there is a major focus on the study of juvenile/adult DR, the effects of hyperglycemia during early retinal development are less well studied. Recent studies in embryonic zebrafish models of nutritional hyperglycemia (high-glucose exposure) have revealed that hyperglycemia leads to decreased cell numbers of mature retinal cell types, which has been related to a modest increase in apoptotic cell death and altered cell differentiation.

Optical Switching Between Long-lived States of Opsin Transmembrane Voltage Sensors.

Opsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better understand the photophysical properties of OTVSs, we used a combination of UV-Vis absorption, fluorescence and FT-Raman spectroscopy to characterize QuasAr2 and NovArch, two closely related mutants derived from the proton pump archaerhodopsin-3 (AR3). We find both QuasAr2 and NovArch can be optically cycled repeatedly between O-like and M-like states using 5-min exposure to red (660 nm) and near-UV (405 nm) light.

Analog Retinal Redshifts Visible Absorption of QuasAr Transmembrane Voltage Sensors into Near-infrared.

Opsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino-modified dimethylphenyl ring results in over a 100-nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively.

Electronic Preresonance Stimulated Raman Scattering Imaging of Red-Shifted Proteorhodopsins: Toward Quantitation of the Membrane Potential.

Voltage imaging allows mapping of the membrane potential in living cells. Yet, current intensity-based imaging approaches are limited to relative membrane potential changes, missing important information conveyed by the absolute value of the membrane voltage. This challenge arises from various factors affecting the signal intensity, such as concentration, illumination intensity, and photobleaching.

Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate.

Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR).

Insight into the chromophore of rhodopsin and its Meta-II photointermediate by F solid-state NMR and chemical shift tensor calculations.

19F nuclei are useful labels in solid-state NMR studies, since their chemical shift and tensor elements are very sensitive to the electrostatic and space-filling properties of their local environment. In this study we have exploited a fluorine substituent, strategically placed at the C-12-position of 11-cis retinal, the chromophore of visual rhodopsins. This label was used to explore the local environment of the chromophore in the ground state of bovine rhodopsin and its active photo-intermediate Meta II.

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K.

A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel "open" state.

Coupled HOOP signature correlates with quantum yield of isorhodopsin and analog pigments.

With a quantum yield of 0.66±0.03 the photoisomerization efficiency of the visual pigment rhodopsin (11-cis⇒all-trans chromophore) is exceptionally high.

Heterologous expression of melanopsin: Present, problems and prospects.

Melanopsin, the photosensory pigment of specialized mammalian retinal ganglion cells, is involved in various non-image forming tasks such as pupillary light reflex, circadian entrainment and irradiance detection. Melanopsin genes have been detected in all vertebrate classes and are resolved in two lineages, Opn4m and Opn4x. In addition, two splice variants have been found in several species leading to a short (OPN4-S) and a long (OPN4-L) isoform, mainly differing in the length of the C terminus.

Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification.

Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs.

Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.

Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1).

Coexpression of three opsins in cone photoreceptors of the salamander Ambystoma tigrinum.

Although more than one type of visual opsin is present in the retina of most vertebrates, it was thought that each type of photoreceptor expresses only one opsin. However, evidence has accumulated that some photoreceptors contain more than one opsin, in many cases as a result of a developmental transition from the expression of one opsin to another. The salamander UV-sensitive (UV) cone is particularly notable because it contains three opsins (Makino and Dodd [1996] J Gen Physiol 108:27-34).

Large scale expression and purification of mouse melanopsin-L in the baculovirus expression system.

Melanopsin is the mammalian photopigment that primarily mediates non-visual photoregulated physiology. So far, this photopigment is poorly characterized with respect to structure and function. Here, we report large-scale production and purification of the intact long isoform of mouse melanopsin (melanopsin-L) using the baculovirus/insect cell expression system.

A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR.

Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-(13)C(2)-retinylidene-rhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (~120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site.

Cyclopropyl and isopropyl derivatives of 11-cis and 9-cis retinals at C-9 and C-13: subtle steric differences with major effects on ligand efficacy in rhodopsin.

Retinal is the natural ligand (chromophore) of the vertebrate rod visual pigment. It occurs in either the 11-cis (rhodopsin) or the 9-cis (isorhodopsin) configuration. In its evolution to a G protein coupled photoreceptor, rhodopsin has acquired exceptional photochemical properties.

Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium.

Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates.

Eye development and retinal differentiation in an altricial fish species, the senegalese sole (Solea senegalensis, Kaup 1858).

We describe the major events in the retinogenesis in an altricial fish species, the Senegalese sole. The major developmental events in the sole retina occurred early after hatching (posthatching day 0, P0). Thus, (1) plexiform layers became recognizable at P1.

Fluoro derivatives of retinal illuminate the decisive role of the C(12)-H element in photoisomerization and rhodopsin activation.

Rhodopsin, the visual pigment of the vertebrate rod cell, is among the best investigated members of the G-protein-coupled receptor family. Within this family a unique characteristic of visual pigments is their covalently bound chromophore, 11-cis retinal, which acts as an inverse agonist. Upon illumination it can be transformed into the all-trans isomer that acts as a full agonist.

A single assay for multiple storage-sensitive red blood cell characteristics by means of infrared spectroscopy.

Background: To maintain a high quality of red blood cells (RBCs), RBC characteristics must be followed during storage under blood bank conditions. By means of infrared (IR) spectroscopy, several characteristics can be measured simultaneously.

Study Design And Methods: IR spectra were acquired for samples from RBCs that were collected and stored according to Dutch blood bank procedures for a period of up to 50 days.

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture.

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (13C, 15N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker's yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines.

Cell differentiation in the retina of an epibenthonic teleost, the Tench (Tinca tinca, Linneo 1758).

Here we present a detailed study of the major events in the retinal histogenesis in a freshwater epibenthonic fish species, the Tench (Tinca tinca, Linneo 1758) during embryonic, prolarval, larval, and juvenile stages, using classical histological and immunohistological methods, providing a complete neurochemical characterization of retinal cells. We find a morphologically undifferentiated retina during embryonic stages and even at the hatching stage (postnatal day 0, P0). However, the emergence of the different retinal layers occurs in the first postnatal day (P1).

Light penetration and photoisomerization in rhodopsin studied by numerical simulations and double-quantum solid-state NMR spectroscopy.

The penetration of light into optically thick samples containing the G-protein-coupled receptor rhodopsin is studied by numerical finite-element simulations and double-quantum solid-state NMR experiments. Illumination with white light leads to the generation of the active bathorhodopsin photostate in the outer layer of the sample but generates a large amount of the side product, isorhodopsin, in the sample interior. The overall yield of bathorhodopsin is improved by using monochromatic 420 nm illumination and by mixing the sample with transparent glass beads.

Towards an interpretation of 13C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor.

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired (13)C chemical shifts along the retinylidene chain of the chromophore.

Monitoring of biomass composition from microbiological sources by means of FT-IR spectroscopy.

An FT-IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability.