[Physiology of the visual retinal signal: From phototransduction to the visual cycle]. / Physiologie du signal visuel rétinien : de la phototransduction jusqu'au cycle visuel.

The retinal photoreceptors (rods and cones) are responsible for light absorption and transduction of the signal, which is transmitted to the other retinal nerve cells and then to the brain. The chromophore of visual pigments of rods and cones is a particular isomer of a vitamin A derivative. Light absorption by this chromophore leads to its isomerization and to a phototransduction cascade, which results in photoreceptor hyperpolarization and cessation of glutamate secretion at their synaptic terminals. Phototransduction of cones and rods differs in their signal amplification and inactivation, which is consistent with their respective functions. The rods serve for dim light vision, whereas color and detailed vision is provided by cones. The rods are thus much more sensitive than cones, but the time course of cones' photoresponse is ∼10 times faster than that of rods. The orientation of cone visual pigments in the retina is optimized to achieve their function. The isomerized chromophore of visual pigments is regenerated by a mechanism known as the visual cycle. This process takes place mainly in the retinal pigment epithelium for the rods and the glial Müller cells for the cones. Mutations of a large number of proteins involved in visual phototransduction and in the retinoid visual cycle are responsible for hereditary diseases leading to photoreceptor degeneration. However, gene therapy offers quite a bit of hope for treatment.

Sedimentation field flow fractionation monitoring of bimodal wheat starch amylolysis.

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this study, we investigated the capacity of sedimentation field flow fractionation (SdFFF) to monitor the amylolysis of a bimodal starch population: native wheat starch. Results demonstrated a correlation between fractogram changes and enzymatic hydrolysis. Furthermore, SdFFF was used to sort sub-populations which enhanced the study of granule size distribution changes occurring during amylolysis. These results show the interest in coupling SdFFF with particle size measurement methods to study complex starch size/density modifications associated to hydrolysis. These results suggested different applications such as the association of SdFFF with structural investigations to better understand the specific mechanisms of amylolysis or starch granule structure.

Sedimentation field flow fractionation monitoring of rice starch amylolysis.

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this work, we investigated the capacity of SdFFF to monitor the native rice starch amylolysis. In order to determine if fractogram changes observed were correlated to granule biophysical modifications which occurred during amylolysis, SdFFF separation was associated with particle size distribution analysis. The results showed that SdFFF is an effective tool to monitor amylolysis of native rice starch. SdFFF analysis was a rapid (less than 10 min), simple and specific method to follow biophysical modifications of starch granules. These results suggested many different applications such as testing series of enzymes and starches. By using sub-population sorting, SdFFF could be also used to better understand starch hydrolysis mechanisms or starch granule structure.

Design of functionalized lipids and evidence for their binding to photosystem II core complex by oxygen evolution measurements, atomic force microscopy, and scanning near-field optical microscopy.

Photosystem II core complex (PSII CC) absorbs light energy and triggers a series of electron transfer reactions by oxidizing water while producing molecular oxygen. Synthetic lipids with different alkyl chains and spacer lengths bearing functionalized headgroups were specifically designed to bind the Q(B) site and to anchor this large photosynthetic complex (240 kDa) in order to attempt two-dimensional crystallization. Among the series of different compounds that have been tested, oxygen evolution measurements have shown that dichlorophenyl urea (DCPU) binds very efficiently to the Q(B) site of PSII CC, and therefore, that moiety has been linked covalently to the headgroup of synthetic lipids. The analysis of the monolayer behavior of these DCPU-lipids has allowed us to select ones bearing long spacers for the anchoring of PSII CC. Oxygen evolution measurements demonstrated that these long-spacer DCPU-lipids specifically bind to PSII CC and inhibit electron transfer. With the use of atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), it was possible to visualize domains of PSII CC bound to DCPU-lipid monolayers. SNOM imaging has enabled us to confirm that domains observed by AFM were composed of PSII CC. Indeed, the SNOM topography images presented similar domains as those observed by AFM, but in addition, it allowed us to determine that these domains are fluorescent. Electron microscopy of these domains, however, has shown that the bound PSII CC was not crystalline.

Probing the transducin nucleotide binding site with GDP analogues.

An affinity study between the G protein of the visual photoreceptor, transducin, and eight different non-hydrolyzable GDP analogues is described. Imidodiphosphate derivatives have been shown to exhibit good affinities to transducin. This very important heterotrimeric G protein is shown to be highly restrictive with regard to structural modifications of the nucleotide at the pyrophosphate moiety, at the 3' position on ribose, as well as at the N1 position of the purine.

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2.

The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.

The interaction between lipid derivatives of colchicine and tubulin: consequences of the interaction of the alkaloid with lipid membranes.

Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by binding tubulin very tightly. Colchicine also possesses anti-inflammatory properties which are not well understood yet. Here we show that colchicine tightly interacts with lipid layers. The physical and biological properties of three different lipid derivatives of colchicine are investigated parallel to those of membrane lipids in the presence of colchicine. Upon insertion in the fatty alkyl chains, colchicine rigidifies the lipid monolayers in a fluid phase and fluidifies rigid monolayers. Similarly X-ray diffraction data show that lecithin-water phases are destabilized by colchicine. In addition, an unexpectedly drastic enhancement of the photoisomerization rate of colchicine into lumicolchicine in the lipid environment is observed and further supports insertion of the alkaloid in membranes. Finally the interaction of colchicine with lipids makes the drug inaccessible to tubulin. The possible in vivo significance of these results is discussed.

Expression of the alpha 5 integrin subunit gene promoter is positively regulated by the extracellular matrix component fibronectin through the transcription factor Sp1 in corneal epithelial cells in vitro.

The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin alpha(5)beta(1). In this work, we determined whether the activity directed by the alpha(5) gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/alpha(5) promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the alpha(5) FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3' half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its alpha(5)beta(1) integrin activates a signal transduction pathway that results in the transcriptional activation of the alpha(5) gene likely through altering the phosphorylation state of Sp1.

Can we produce a human corneal equivalent by tissue engineering?

Tissue engineering is progressing rapidly. Bioengineered substitutes are already available for experimental applications and some clinical purposes such as skin replacement. This review focuses on the development of reconstructed human cornea in vitro by tissue engineering. Key elements to consider in the corneal reconstruction, such as the source for epithelial cells and keratocytes, are discussed and the various steps of production are presented. Since one application of this human model is to obtain a better understanding of corneal wound healing, the mechanisms of this phenomenon as well as the function played both by membrane-bound integrins and components from the extracellular matrix have also been addressed. The analysis of integrins by immunohistofluorescence labelling of our reconstructed human cornea revealed that beta(1), alpha(3), alpha(5), and alpha(6) integrin subunits were expressed but alpha(4) was not. Laminin, type VII collagen and fibronectin were also detected. Finally, the future challenges of corneal reconstruction by tissue engineering are discussed and the tremendous applications of such tissue produced in vitro for experimental as well as clinical purposes are considered.

Monitoring of phospholipid monolayer hydrolysis by phospholipase A2 by use of polarization-modulated Fourier transform infrared spectroscopy.

Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) was used to follow the hydrolysis of phospholipid monolayers at the air-water interface by phospholipase A2 (PLA2). The decrease in the intensity of the nuC=O ester band of dipalmitoylphosphatidylcholine at 1733 cm(-1) and the appearance of two new infrared bands in the 1530-1580 cm(-1) region allowed to monitor phospholipid hydrolysis by PLA2. Indeed, the decrease in the intensity of the band at 1733 cm(-1) was attributed to the enzymatic hydrolysis of the acyl ester linkage of the sn-2 fatty acid on the glycerol backbone whereas the doublet appearing at 1537 and 1575 cm(-1) was attributed to the nu(a) COO- vibration of the newly formed calcium-palmitate. The presence of this band as a doublet indicates the formation of a crystalline-like calcium-palmitate monolayer. This observation supports our previously postulated mechanism for the formation of PLA2 domains at the air-water interface. Definitive assignment of the infrared bands has been possible by measuring PM-IRRAS spectra of the individual hydrolysis products (palmitic acid and lysopalmitoylphosphatidylcholine) as well as of 1-caproyl-2-palmitoyl-phosphatidylcholine and 1-palmitoyl-2-caproylphosphatidylcholine monolayers before and after hydrolysis by PLA2.

Transcriptional regulation of the alpha 4 integrin subunit gene in the metastatic spread of uveal melanoma.

Recently, expression of the alpha 4 integrin subunit has been shown to be inversely correlated with the invasive potential of B16 mouse epidermal melanoma. The purpose of this study was to establish whether expression of the human alpha 4 integrin subunit gene might be similarly regulated in human uveal melanoma which has varying degrees of invasiveness, and whether such modifications are determined by alterations in the transcriptional activity directed by the alpha 4 gene promoter. Two metastatic variants (MH5 and MH10) derived from a human uveal melanoma (SP6.5) were used. Expression studies were performed by transiently transfecting each of these cell lines with recombinant plasmids bearing various lengths of the alpha 4 promoter fused to the CAT reporter gene, and were further validated by Northern blot analyses of the alpha 4 transcript. Both transient transfection and mRNA analyses provided evidence that the transcriptional activity directed by the alpha 4 promoter sequences extending up to position -76 and -120 was indeed inversely correlated to the potential of uveal melanoma to yield metastasis. Experiments in electrophoretic mobility shift assay (EMSA) demonstrated that binding of the nuclear proteins that likely account for transcription of the alpha 4 gene to alpha 4.1 (namely Bp1, Bp2, Bp4, and Bp5) was dramatically reduced in uveal melanoma, but not in normal uveal melanocytes. These results highlight the fundamental function the alpha 4 integrin subunit may play in the ability of tumor cells to evade the primary tumor and form metastasis.

Polarization-modulated infrared spectroscopy and x-ray reflectivity of photosystem II core complex at the gas-water interface.

The state of photosystem II core complex (PS II CC) in monolayer at the gas-water interface was investigated using in situ polarization-modulated infrared reflection absorption spectroscopy and x-ray reflectivity techniques. Two approaches for preparing and manipulating the monolayers were examined and compared. In the first, PS II CC was compressed immediately after spreading at an initial surface pressure of 5.7 mN/m, whereas in the second, the monolayer was incubated for 30 min at an initial surface pressure of 0.6 mN/m before compression. In the first approach, the protein complex maintained its native alpha-helical conformation upon compression, and the secondary structure of PS II CC was found to be stable for 2 h. The second approach resulted in films showing stable surface pressure below 30 mN/m and the presence of large amounts of beta-sheets, which indicated denaturation of PS II CC. Above 30 mN/m, those films suffered surface pressure instability, which had to be compensated by continuous compression. This instability was correlated with the formation of new alpha-helices in the film. Measurements at 4 degreesC strongly reduced denaturation of PS II CC. The x-ray reflectivity studies indicated that the spread film consists of a single protein layer at the gas-water interface. Altogether, this study provides direct structural and molecular information on membrane proteins when spread in monolayers at the gas-water interface.

Polymorphism of the 1-palmitoyl-2-arachidonoyl-phosphatidyl-ethanolamine/dimyristoyl-phos phatidylmethanol mixture, a phospholipase A2 substrate.

The polymorphism of the equimolar mixture of 1-palmitoyl-2-arachidonoyl-phosphatidylethanolamine (PAPE) and dimyristoyl-phosphatidylmethanol (DMPM) was examined by infrared and 31P nuclear magnetic resonance spectroscopy to determine the suitability of this widely used but yet uncharacterized lipid mixture as a phospholipase A2 substrate. The results show that the mixture undergoes a gel-to-liquid crystalline phase transition between 12 and 35 degrees C. The transitions of the individual lipids were also examined. We report that the temperature of the gel-to-liquid crystalline phase transition of DMPM is 40 degrees C. At 2 degrees C, PAPE exists in the fluid lamellar form. This lipid undergoes a lamellar-to-inverted hexagonal phase transition at 31 degrees C. In conclusion, the equimolar PAPE/DMPM mixture forms fluid lamellar phase at the physiological temperature. However, the upper end of the gel-to-liquid crystalline phase transition of the mixture is really close to the physiological temperature and this situation is a serious source of potential artefacts.

Determination of the depth of penetration of the alpha subunit of retinal G protein in membranes: a spectroscopic study.

This paper reports the fluorescence quenching of the alpha subunit of retinal rod outer segment G protein (Gtalpha) by vesicles of brominated phospholipids. Two different brominated phospholipids with the bromine quencher groups attached at the 6-7 and 9-10 positions in one of the fatty acyl chains have been used to estimate the depth of penetration of the Gtalpha protein in the lipid vesicles using steady-state fluorescence quenching techniques. Our studies provide evidence of the interaction between Gtalpha protein, in its active conformation, with the lipid vesicles mimicking natural membranes. This study demonstrates that in vitro the distance between fluorescent tryptophan site of Gtalpha and the membrane surface is approximately 6.5 A.

Phospholipases A2 of rod outer segment-free bovine retinae are different from well-known phospholipases A2.

We have recently demonstrated the presence of phospholipase A2 (PLA2) activity in a rod outer segment-free retinal fraction which we called P200 and which contains neuronal cells, Müller cells and rod inner segments. We report here our results on the characterization of this P200-PLA2 activity. We show that P200 probably contains more than one type of PLA2, as indicated by the results obtained with different chromatographically eluted PLA2-active fractions which were treated with either Ca2+, EGTA, dithiothreitol (DTT) or p-bromophenacyl bromide (pBPB), or heated. Moreover, the results from PLA2 assays using different substrates, as well as those obtained after treatment of the homogenate with H2SO4, guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) and ATP, suggest that P200-PLA2 are different from well-known secretory PLA2, cytosolic PLA2 and Ca2+-independent PLA2. Control experiments using our 'back-and-forth'-thin layer chromatography (bf-TLC) technique allowed us to confirm that, in our assay conditions, the release of fatty acids was due to PLA2 enzymes. These results, which constitute the first characterization of PLA2 of the neural retina, thus suggest that it contains novel types of PLA2 enzyme, in contrast to well-known PLA2.

Bovine retinal pigment epithelium contains novel types of phospholipase A2.

We have recently demonstrated the presence of phospholipase A2 (PLA2) activity in cells from bovine retinal pigment epithelium (RPE) [Jacob et al. (1996) J. Biol. Chem. 271, 19209-19218]. We report here our results on the characterization of this RPE-PLA2 activity. We show that RPE probably contains two types of PLA2 enzyme, as indicated by the results obtained with different PLA2-active fractions eluted from cation-exchange columns and treated with Ca2+/EGTA, dithiothreitol, p-bromophenacyl bromide or heat. These results, in addition to those from PLA2 assays using different substrates, also suggest that RPE-PLA2 enzymes are different from the well-known secretory, cytoplasmic and Ca2+-independent forms. Sequential extraction of RPE with (1) isotonic, (2) hypertonic and (3) detergent-containing PBS argues for the presence of weakly membrane-associated enzymes. Control experiments using 'back and forth' TLC allowed us to discriminate between PLA2 and phospholipase C/diacylglycerol lipase activity and confirmed that, in our assay conditions, the release of fatty acids was indeed due to PLA2 enzymes. These results, together with those obtained by treating RPE homogenates with H2SO4, guanosine 5'-[gamma-thio]triphosphate, ATP and different protease inhibitors, permitted us to make the first characterization of these RPE-PLA2 enzymes. We conclude that RPE contains novel types of PLA2 that are different from the secretory, cytoplasmic and Ca2+-independent forms.

Membrane microstructural templates for enzyme domain formation.

Soluble proteins can spontaneously self-organize into two-dimensional domains at membrane interfaces, given sufficient mobility and specificity to membrane-localized ligands. The authors' recent results studying interfacial domain formation of the membrane-active enzyme, phospholipase A2, indicate that lateral phase separation of heterogeneous membrane mixtures creates anionic templates of specific morphology onto which the enzyme deposits, forming large protein assemblies. Selective removal of membrane components (lysolipid or fatty acid) produces different enzyme interfacial responses and domain morphologies. This leads to the conclusion that complex chemical and physical interactions laterally in the lipid membrane interface as well as between bound protein molecules play a role in organizing protein structures.

Presence of a light-independent phospholipase A2 in bovine retina but not in rod outer segments.

Rod outer segments (ROS) are responsible for the visual transduction process. Rhodopsin, which constitutes 85-90% of ROS proteins, absorbs light photons, changes its conformation, and then binds to a heterotrimeric G-protein called transducin. As a consequence, transducin dissociates into Talpha and Tbetagamma subunits. The presence in ROS of a phospholipase A2 (PLA2) stimulated by light and guanosine 5'-O-(3-thio)triphosphate was first demonstrated in 1987 (Jelsema, C. L.(1987) J. Biol. Chem. 262, 163-168). This led that author to conclude that ROS PLA2 could be involved in the phototransduction process, and raised the possibility of receptor-mediated activation of PLA2 via G-proteins in cell types other than rods. However, the biochemical characteristics and the role of this PLA2 have not been fully elucidated. We have tried to reproduce some of the results previously reported in order to further characterize this enzyme. We have found that, in our hands, there is neither light-dependent nor GTP-dependent PLA2 activity in intact purified ROS. We also failed to detect PLA1 activity in those ROS preparations. Nevertheless, we detected significant amounts of PLA2 activity in two subretinal fractions adjacent to ROS: RPE (enriched with retinal pigment epithelial cells) and P200 (presumably containing neuronal cells, Müller cells, and rod inner segments). The enzyme present both in RPE and P200 is light- and GTP-independent, Ca2+- and Mg2+-independent, and seems to be optimally active in the alkaline pH range. Our results suggest that there is, if any, vanishingly little PLA2 or PLA1 activity in intact purified ROS and that the activity levels previously reported in the literature could have been due to a contamination by either RPE or P200. This is supported by our observation that some contaminated ROS preparations were "PLA2 active."

Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy.

The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation.

Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface.

Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.