[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.

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.

Interactions in mixed monolayers between distearoyl-L-phosphatidylethanolamine, rod outer segment phosphatidylethanolamine and all-trans retinal. Effect of pH.

The interactions in mixed monolayers between distearoyl-L-phosphatidylethanolamine, natural phosphatidylethanolamine purified from bovine rod outer segments and all-trans retinal have been studied at the nitrogen/water interface at 21.0 +/- 0.5 degrees C. Seven mixtures of each phospholipid with all-trans retinal, covering the whole range of molar fractions, were studied. The monolayers were spread on a 1 X 10(-3) M phosphate buffer subphase at three different pH values, 5.5, 7.1 and 8.2. The results for the two series of mixtures are strikingly different. The surface phase rule shows that all-trans retinal is miscible with the natural phospholipid at the interface. Small, negative deviations with respect to the additivity rule are observed in this case. The excess free energies of mixing were also calculated as a function of concentration for this system at four different surface pressures, 5, 7, 10 and 13 mN X m-1. They are negative for the four surface pressures considered and symmetrical with respect to the mole fraction. On the other hand, when distearoyl-L-phosphatidylethanolamine is mixed with all-trans retinal, the components are no longer miscible at the interface. This marked difference in behaviour between the two lipids reflects the importance of hydrophobic interactions in the mixed monolayers of phospholipids with retinals. Furthermore, for the two series of mixtures, the surface pressure isotherms do not show any significant shift when the subphase pH is changed from 5.5 to 8.2. This behaviour raises questions about the formation of a Schiff base between phosphatidylethanolamine and retinal at the interface. It is suggested that, owing to the nature of the disk membranes, such an effect would also be observed in vivo. The possible implications of this are discussed, particularly with respect to questions pertaining to the stability of the retinal chromophore.

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.

Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy.

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.

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.

Mixed monolayers of natural and polymeric phospholipids: structural characterization by physical and enzymatic methods.

This study has focused on physical characterization and enzymatic hydrolysis of mixed monolayers of a natural phospholipid substrate and a polymerizable phospholipid analogue. Such a mixed system presents the possibility to stabilize model biomembranes, vary the molecular environment within the layer through polymerization and simultaneously examine these influences on monolayer structure. Phospholipase A2 was used here as a sensitive probe of the molecular environment within these mixed, polymerizable monolayers to complement information obtained from isotherm and isobar data. The results clearly show a strong influence of molecular environment on phospholipase A2 activity, even if differences in the physical state of mixed monolayers are not detectable with isotherm and isobar measurements. Physical characterization indicated that both monomeric and polymeric mixed monolayers were phase-mixed. Enzyme hydrolysis, however, showed large differences in the ability of the enzyme to selectively hydrolyze the natural phosphatidylcholine component from the monomeric as opposed to the polymeric mixtures. This demonstrates a high sensitivity of phospholipase A2 to distinguish subtle differences in molecular arrangement within mixed monolayers on a molecular level.

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.

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.

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.

Multiple regulatory elements control the basal promoter activity of the human alpha 4 integrin gene.

It has been suggested that expression of the genes encoding the alpha 4/beta 1 integrin increases during wound healing of the cornea. As a first step in understanding the mechanisms required to stimulate alpha 4 gene expression during this process, we defined the minimal upstream sequence required to direct basal promoter activity for this gene. Using deletion analyses of the alpha 4 gene upstream sequence, we identified two functionally important negative regulatory elements. Dimethylsulfate (DMS) methylation interference assays provided evidence for the binding of a single nuclear protein to tandemly repeated homologous cis-acting elements (designated alpha 4.1 and alpha 4.2) from the alpha 4 basal promoter that share the core sequence 5'-GTGGGT-3'. The formation of a protection only at alpha 4.1 in DNase I footprinting suggested that it is the primary target element for the binding of nuclear proteins. Three distinct nuclear proteins bound a double-stranded oligonucleotide bearing the DNA sequence of alpha 4.1 to produce specific DNA-protein complexes (R1 to R3) in gel-shift assays, from which that producing R3 was identified as the protein yielding DNase I protection at alpha 4.1. Detailed mutational analysis of alpha 4.1 and alpha 4.2 indicated that both elements positively regulate gene expression in primary cultures of corneal epithelial cells and Jurkat tissue culture cells, which is consistent with the deletion analysis. However, when transiently transfected into pituitary GH4C1, the alpha 4.2 mutants yielded increased chloramphenicol acetyl transferase activity therefore demonstrating that these elements have the ability to function either as positive or negative regulators of gene transcription in a manner that is dependent on the type of cell transfected.

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.

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.

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.

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.

Direct evidence for the formation of a monolayer from a bilayer. An ellipsometric study at the nitrogen-water interface.

Direct evidence for the formation of a monolayer from a bilayer was measured by ellipsometry after spreading unilamellar vesicles of dioleoyl phosphatidylcholine (DOPC) at the nitrogen-water interface. The ellipsometric isotherms of DOPC vesicles and DOPC spread from an organic solvent were compared and found similar. From the observed ellipsometric angle (delta delta) in the plateau region (-1.04 degrees) and literature data for refractive indices of an anisotropic film similar to DOPC, we have calculated a thickness of 20 +/- 1 A. These results strongly suggest that, similarly to DOPC spread from an organic solvent, DOPC vesicles form a monolayer when spread at the nitrogen-water interface.

The binding of G-protein to rod outer segment phospholipids at the nitrogen-water interface.

In the visual process, one photoexcited rhodopsin (R*) catalyzes the activation of hundreds of G-proteins. It remains to be determined whether G-protein and R* find one another by membrane surface diffusion of these components (diffusion model) or by diffusion of G-protein through the aqueous phase (hopping model). A monolayer of each main rod outer segment (ROS) phospholipid interacting with a subphase containing G-protein, has been used to simulate the interaction of G-protein with the cytoplasmic surface of discal membranes. The possible diffusion of G-protein through the aqueous phase was then measured by observing its adsorption-desorption in the monolayer of each main ROS phospholipid. From examination of surface pressure and ellipsometric isotherms at the nitrogen-water interface, we have determined that once incorporated into the monolayer, the G-protein remains associated, independent of surface pressure, thus providing evidence against the hopping model.

Interaction between biotin lipids and streptavidin in monolayers: formation of oriented two-dimensional protein domains induced by surface recognition.

Highly specific ligand-receptor interactions generally characterize surface recognition reactions. Such processes can be simulated by streptavidin-biotin-specific binding. Biotin lipids have thus been synthesized, and their interaction with streptavidin (or avidin) at the air-water interface was directly shown by measurement of surface pressure isotherms and fluorescence microscopy. These proteins interact with the biotin lipid monolayer via specific binding or nonspecific adsorption. Both phenomena were clearly distinguished by use of the inactivated form of streptavidin. The binding of fluorescein-labeled streptavidin to monolayers was also directly observed by fluorescence microscopy. The fluorescence of the protein domains is directly related to the state of polarization of the exciting light. This anisotropy can only be explained by the formation of oriented two-dimensional biotin lipid-streptavidin domains.