Catabolism of myo-Inositol to Precursors Utilized for De Novo Glycerolipid Biosynthesis

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.     

Transbilayer distribution of phospholipids in photoreceptor membrane studied with trinitrobenzenesulfonate alone and in combination with phospholipase D

In a further study of the transbilayer distribution of phospholipids in rod disk membranes, the amino group reagent, trinitrobenzenesulfonate, and the phospholipid-hydrolyzing enzyme, phospholipase D, have been used alone and in combination.Under carefully defined conditions (1 mM trinitrobenzenesulfonate, pH 7.4, 20°C, darkness), trinitrobenzenesulfonate yields limited final levels of modification of phosphatidylethanolamine and phosphatidylserine, suggesting only minor reagent penetration and membrane disturbance under these conditions.Treatment of stacked disks with trinitrobenzenesulfonate under these conditions leads to a biphasic modification of the a aminophospholipids. Relatively fast (less than 1 h) modification of 50% phosphatidylethanolamine and 40% phosphatidylserine occurs, slowly rising (approx. 3 h) to 60 and 50%, respectively.Extensive treatment of stacked disks with phospholipase D leads to the hydrolysis of 55% phosphatidylcholine and 50% phosphatidylethanolamine, while phosphatidylserine is hardly attacked by this enzyme.Treatment of stacked disks with trinitrobenzenesulfonate after prior treatment with phospholipase D leads to no further modification than that maximally obtained with either reagent alone: about one-half of the three major phospholipid classes is accessible. Although both reagents differ greatly in molecular size, mode of action and other properties, they apparently see the same pool of phosphatidylethanolamine, their joint substrate. Considering that we start with the original right-side-out configuration, that all phospholipids can in principle be modified (no shielding) and that the membrane remains essentially intact, we conclude that the accessible lipid pool represents the outer face of the disk membranes.These results confirm our earlier conclusions from treatment with three phospholipases that the three major phospholipids are nearly symmetrically distributed over the two faces of the disk membrane.The divergence with the conclusions of other investigators is most likely explained by their use of disk membranes (disk vesicles) in which the original phospholipid distribution had not been maintained and/or of conditions under which trinitrobenzenesulfonate markedly penetrates the membrane.     

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

Developmentally Regulated Linker Histone H1c Promotes Heterochromatin Condensation and Mediates Structural Integrity of Rod Photoreceptors in Mouse Retina

Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H1⁰ triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.     

Pharmacological characterization of the effects of taurine on calcium uptake in the rat retina

Summary Taurine is known to increase ATP-dependent calcium ion (Ca²⁺) uptake in retinal membrane preparations and in isolated rod outer segments (ROS) under low calcium conditions (10M) (Pasantes-Morales and Ordóñez, 1982; Lombardini, 1991). In this report, ATP-dependent Ca²⁺ uptake in retinal membrane preparations was found to be inhibited by 5M cadmium (Cd²⁺), suggesting the involvement of cation channel activation. The activation of cGMP-gated cation channels, which are found in the ROS, is a crucial step in the phototransduction process. An inhibitor of cGMP-gated channels, LY83583, was found to inhibit taurine-stimulated ATP-dependent Ca²⁺ uptake but had no effect on ATP-dependent Ca²⁺ uptake in the absence of taurine, indicating that taurine may be increasing ATP-dependent Ca²⁺ uptake through a mechanism of action involving the opening of cGMP-gated channels. The activation of cGMP-gated channels with dibutyryl-cGMP and with phosphodiesterase inhibition using zaprinast caused an increase in ATP-dependent Ca²⁺ uptake in isolated ROS, but not in taurine-stimulated ATP-dependent Ca²⁺ uptake. LY83583 had the same effects in isolated ROS as in retinal membrane preparations. Another inhibitor of cGMP-gated channels, Rp-8-Br-PET-cGMPS, produced the same pattern of inhibition in isolated ROS as LY83583. Thus, there appears to be a causal link between taurine and the activation of the cGMP-gated channels in the ROS under conditions of low calcium concentration, a connection that suggests an important role for taurine in the visual signalling function of the retina.     

Synaptic connections of cone bipolar cells that express the neurokinin 1 receptor in the rabbit retina

We have investigated and further characterized, in the rabbit retina, the synaptic connectivity of the ON-type cone bipolar cells that are immunoreactive for an antibody against the neurokinin-1 receptor (NK1R). NK1R-immunoreactive bipolar cell axons terminate in stratum 4 of the inner plexiform layer. The axons of NK1R-positive bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and from putative AII amacrine cells via gap junctions. The major outputs from NK1R-positive bipolar cells make contacts with amacrine cell processes. The most frequent postsynaptic dyads comprise two amacrine cell processes. Double-labeling experiments with antibodies against NK1R and either calretinin or glycine have demonstrated that NK1R-immunoreactive bipolar cells form gap junctions with AII amacrine cells. Thus, NK1R-positive cone bipolar cells, together with calbindin-positive cone bipolar cells, may play an important role in transferring rod signals to the ON-type ganglion cells of the cone pathway in the rabbit retina.I.-B. Kim and M.R. Park contributed equally to this work.This work was supported by the Ministry of Science and Technology of Korea (grant no. M1-0108-00-0059; Neurobiology Support Grant).     

Investigation on chlorosomal antenna geometries: tube,lamella and spiral-type self-aggregates

Molecular mechanics calculations and exciton theory have been used to study pigment organization in chlorosomes of green bacteria. Single and double rod, multiple concentric rod, lamella, and Archimedean spiral macrostructures of bacteriochlorophyll c molecules were created and their spectral properties evaluated. The effects of length, width, diameter, and curvature of the macrostructures as well as orientations of monomeric transition dipole moment vectors on the spectral properties of the aggregates were studied. Calculated absorption, linear dichroism, and polarization dependent fluorescence-excitation spectra of the studied long macrostructures were practically identical, but circular dichroism spectra turned out to be very sensitive to geometry and monomeric transition dipole moment orientations of the aggregates. The simulations for long multiple rod and spiral-type macrostructures, observed in recent high-resolution electron microscopy images (Oostergetel et al., FEBS Lett 581:5435–5439, 2007) gave shapes of circular dichroism spectra observed experimentally for chlorosomes. It was shown that the ratio of total circular dichroism intensity to integrated absorption of the Q _y transition is a good measure of degree of tubular structures in the chlorosomes. Calculations suggest that the broad Q _y line width of chlorosomes of sulfur bacteria could be due to (1) different orientations of the transition moment vectors in multi-walled rod structures or (2) a variety of Bchl-aggregate structures in the chlorosomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Involvement of Lysophosphatidic Acid,Sphingosine 1-Phosphate and Ceramide 1-Phosphate in the Metabolization of Phosphatidic Acid by Lipid Phosphate Phosphatases in Bovine Rod Outer Segments

The aim of the present research was to evaluate the generation of [2-3H]diacylglycerol ([2-3H]DAG) from [2-3H]-Phosphatidic acid ([2-3H]PA) by lipid phosphate phosphatases (LPPs) at different concentrations of lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) in purified ROS obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. Western blot analysis revealed the presence of LPP3 exclusively in all membrane preparations. Immunoblots of entire ROS and depleted ROS did not show dark-light differences in LPP3 levels. LPPs activities were diminished by 53% in BLROS with respect to DROS. The major competitive effect on PA hydrolysis was exerted by LPA and S1P in DROS and by C1P in BLROS. LPPs activities in depleted ROS were similar to the activity observed in entire DROS and BLROS, respectively. LPA, S1P and C1P competed at different extent in depleted DROS and BLROS. Sphingosine and ceramide inhibited LPPs activities in entire and depleted DROS. Ceramide also inhibited LPPs activities in entire and in depleted BLROS. Our findings are indicative of a different degree of competition between PA and LPA, S1P and C1P by LPPs depending on the illumination state of the retina.