Enzymatic reduction of 11-cis-retinal bound to cellular retinal-binding protein

Cellular retinal-binding protein from bovine retina purifies with bound 11-cis-retinal and 11-cis-retinol as endogenous ligands. Inasmuch as these retinoids are interconvertible by a dehydrogenase reaction the accessibility of the aldehyde function of bound 11-cis-retinal to chemical and enzymatic reducing agents was determined. An 11-cis-retinol dehydrogenase from retinal pigment epithelial microsomes, first described by Lion, F., Rotmans, J.P., Daemen, F.J.M. and Bonting, S.L. (Biochim. Biophys. Acta 384, 283–292, 1975) was found to reduce complexed 11-cis-retinal at pH 5.5 and 37°C rapidaly and nearly quantitatively. The product of the reduction, 11-cis-retinol, remained complexed with the binding protein following the reaction. Reduction proceeded 3-times more rapidly with NADH than with NADPH. No change in geometrical isomeric configuration occurred during the reaction. The dehydrogenase from retinal pigment epithelium oxidized 11-cis-retinol complexed with cellular retinal-binding protein at pH 8.5 in the presence of NAD. In spite of the ready enzymatic reduction of 11-cis-retinal complexed with cellular retinal-binding protein, the aldehyde function was inaccessible to several chemical reducing agents. Incubation of the complex with NaBH₄ at pH 7.5 and NaCNBH₃ or borane dimethylamine at pH 5.5 did not result in reduction of 11-cis-retinal unless the complex had been exposed to white light, a treatment known to produce all-rans-retinal which has little affinity for the binding protein. Liver alcohol dehydrogenase produced only 10% reduction of 11-cis-retinal complexed with cellular retinal-binding protein in 15 min at 37°C when added in amounts which produced about 60% reduction of the uncomplexed retinoid. The results suggest that the interaction between the 11-cis-retinol dehydrogenase and the 11-cis-retinal complexed to cellular retinal-binding protein is a specific one of that the binding protein may function as a substrate carrier for a dehydrogenase.     

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.The retinoid cycle is a fundamental metabolic process in the vertebrate retina responsible for continuous generation of 11-cis-retinal from its all-trans-isomer (1-3). Because 11-cis-retinal is the chromophore of rhodopsin and cone visual pigments (4), disabling mutations in genes encoding proteins of the retinoid cycle can cause a spectrum of retinal diseases affecting sight (3). Moreover, the efficiency of the mammalian visual system and health of photoreceptors and retinal pigment epithelium (RPE)² decrease significantly with age. Even in the presence of a functional retinoid cycle, A2E, retinal dimer (RALdi), and other toxic all-trans-retinal condensation products (5-7) can accumulate as a consequence of aging (8). Under experimental conditions, these compounds can produce toxic effects on RPE cells (9-11). Patients affected by age-related macular degeneration, Stargardt disease, or other retinal diseases associated with accumulation of surrogate markers, such as A2E, all develop retinal degeneration (12). Thus, elucidating the fundamental causes of these age-dependent changes is of increasing importance. Encouragingly, our understanding of both retinoid metabolism outside the eye and production of 11-cis-retinal unique to the eye has accelerated recently (Scheme 1) (1-3), and genetic mouse models are readily available to study these processes and their potential aberrations in vivo (13). Thus, a central question can be addressed, namely what initiates the death of photoreceptor cells and the underlining RPE?Open in a separate windowSCHEME 1.Retinoid flow and all-trans-retinal clearance in the visual cycle. After diffusion from the RPE, the visual chromophore, 11-cis-retinal, combines with rhodopsin and then is photoisomerized to all-trans-retinal. Most of the all-trans-retinal dissociates from opsin into the cytoplasm, where it is reduced to all-trans-retinol by RDHs, including RDH8. The fraction of all-trans-retinal that dissociates into the disc lumen is transported by ABCA4 into the cytoplasm (23) before it is reduced. All-trans-retinol then is translocated to the RPE, esterified by LRAT, and recycled back to 11-cis-retinal. Mutations of ABCA4 are associated with human macular degeneration, Stargardt disease, and age-related macular degeneration (55, 56).Several mechanisms associated with retinoid metabolism may contribute to different retinopathies (1). For example, lack of retinoids in LRAT (lecithin:retinol acyltransferase) or chromophore in retinoid isomerase knock-out (Rpe65^-/-) mice leads to rapid degeneration of cone photoreceptors and slowly progressive death of rods (14). Such mice do not produce toxic condensation products from all-trans-retinal. Instead, their retinopathies have been attributed to continuous activation of visual phototransduction (15) due to either the basal activity of opsin (16-18) or disordered vectorial transport of cone visual pigments without bound chromophore (19). Paradoxically, an abnormally high flux of retinoids through the retinoid cycle can also lead to retinopathy in other mouse models (20, 21). Animal models featuring anomalies in the retinoid cycle illustrate the importance of chromophore regeneration and provide an approach to elucidating mechanisms involved in human retinal dysfunction and disease.Recently, we showed that mice carrying a double knock-out of Rdh8 (retinol dehydrogenase 8), one of the main enzymes that reduces all-trans-retinal in rod and cone outer segments (22), and Abca4 (ATP-binding cassette transporter 4), which transports all-trans-retinal from the inside to the outside of disc membranes (23), rapidly accumulate all-trans-retinal condensation products and exhibit accentuated RPE/photoreceptor dystrophy at an early age (24). Although these studies suggest retinoid toxicity, it is still unclear if the elevated levels of retinal and/or its condensation products, such as A2E, are the cause of this retinopathy or merely a nonspecific reflection of impaired retinoid metabolism. Here, we report that spent chromophore, all-trans-retinal, is most likely responsible for photoreceptor degeneration in Rdh8^-/-Abca4^-/- mice. Toxic effects of all-trans-retinal include caspase activation and mitochondria-associated cell death.     

Specific photoisomerization of retinal in squid rhodopsin and metarhodopsin

The composition of retinal isomers in the photosteady-state mixtures formed from squid rhodopsin and metarhodopsin was determined by high-pressure liquid chromatography. A large amount of 9-cis-retinal was obtained at liquid N₂ temperature when rhodopsin was irradiated with orange light, but only small quantities of 9-cis-retinal were obtained at 15°C. Scarcely any 9-cis-retinal was produced from metarhodopsin by irradiation at liquid N₂ temperature. A large quantity of 7-cis-retinal was found in the photoproduct of rhodopsin irradiated at solid carbon dioxide temperature, but not at 15°C and liquid N₂ temperature. 7-cis-Retinal was not produced from metarhodopsin at any temperatures. These results indicate that the photoisomerization of retinal is regulated by the structure of the retinal-binding site of this protein. The formation of 9-cis- and 7-cis-retinals is forbidden in the metarhodopsin protein.     

Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) drive protein translation in cells and hence these are essential enzymes across life. Inhibition of these enzymes can halt growth of an organism by stalling protein translation. Therefore, small molecule targeting of aaRS active sites is an attractive avenue from the perspective of developing anti-infectives. Febrifugine and its derivatives like halofuginone (HF) are known to inhibit prolyl-tRNA synthetase of malaria parasite Plasmodium falciparum. Here, we present functional and crystallographic data on P. falciparum prolyl-tRNA synthetase (PfPRS). Using immunofluorescence data, we show that PfPRS is exclusively resident in the parasite cytoplasm within asexual blood stage parasites. The inhibitor HF interacts strongly with PfPRS in a non-competitive binding mode in presence or absence of ATP analog. Intriguingly, the two monomers that constitute dimeric PfPRS display significantly different conformations in their active site regions. The structural analyses presented here provide a framework for development of febrifugine derivatives that can seed development of new anti-malarials.     

Molecular charcterization of tatD DNAse gene from Ralstonia paucula RA4T soil bacterium

Ralstonia paucula strain RA4^T, a gram negative, non-spore forming, motile bacterium having positive catalase and oxidase test, was isolated from surface soil. Twin arginine translocation protein type D (TatD) is shown to be located in cytoplasm and exhibits magnesium-dependent DNase. A tatD DNase gene was isolated and cloned from Ralstonia paucula RA4^T genome. Nucleotide sequence analysis of the gene revealed 813 nucleotides encoding a protein of 270 amino acid residues. The tatD gene showed a high similarity to homolog gene from Ralstonia pickettii strain 12D. The deduced polypeptide sequence of TatD DNase from R. paucula RA4^T had a typical catalytic site, HHPLDEHRHDP, and its calculated molecular mass and predicted isoelectric point were 29616 Da and 5.33, respectively. The deduced amino acid sequence showed a high degree of similarity to TatD DNase isoforms from Ralstonia genus and other sources. Predicted three-dimensional structure of TatD confirmed the presence of active site and theoretical function as DNase.     

Denaturation of the high molecular weight protein fraction of mustard (Brassica juncea) and rapeseed (Brassica campestris) by urea or guanidinium hydrochloride

Urea and guanidinium hydrochloride dissociate the 12S protein of mustard and rapeseed to 1.8 S protein and the extent of dissociation depends on the concentration of the denaturant. Mustard (Brassica juncea) protein is more readily dissociated than the rapeseed (Brassica campestris) protein. The reagents denature the protein as evidenced by increase in viscosity, appearance of difference spectra and quenching of fluorescence. Rapeseed protein is denatured more readily than the mustard protein. Analysis of visctosity, spectral and fluoresence data suggests that the first event in the denaturation reaction is the perturbation of the aromatic amino acid residues followed by their exposure to the solvent medium and unfolding of the protein molecule.     

Isolation and characterization of the four major cysteine-proteinase components of the latex of carica papaya L. reactivity characteristics towards 2,2′-dipyridyl disulfide of the thiol groups of papain,chymopapains A and B,and papaya peptidase A

High-quality spray-dried latex of Carica papaya L was fractionated by using SP-Sephadex-C50. The four major cysteine-proteinase components—papain(E.C.3.4.22.2), chymopapains A and B(jointly designated currently as E.C.3.4.22.6), and papaya peptidase A—were isolated and characterized by protein chemical methods and by study of their thiol groups using2,2′-dipyridyl disulfide as a two-protonic-state titrant and reactivity probe. Papain and papaya peptidase A each contain one thiol group/molecule, which in each case is part of the catalytic site, as evidenced by high reactivity toward2,2′-dipyridyl disulfide in acidic media. Chymopapains A and B each contain two thiol groups/molecule, only one of which is essential for catalytic activity. The reactivities of the thiol groups of these enzymes toward2,2′-dipyridyl disulfide at pH4 and10 and activity loss analysis by Tsou Chen-Lu plots each provides a ready means of distinguishing among the four cysteine proteinases. The nonessential thiol groups of chymopapains A and B readily undergo irreversible oxidation. The reactivity characteristics of the essential thiol groups of the four enzymes suggest the presence of somewhat similar interactive cysteine-histidine catalytic center systems in papain, papaya peptidase A, and chymopapain B but a different type of catalytic center environment in chymopapain A.     

Identification of Stringent Response-Related and Potential Serological Proteins Released from Bacillus anthracis Overexpressing the RelA/SpoT Homolog,Rsh Bant

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh _Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh _Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh _Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh _Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh _Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.     

Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus

Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5′-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.     

Crystal structure of the eukaryotic translation initiation factor 2A from Schizosaccharomyces pombe

The eukaryotic translation initiation factor 2A (eIF2A) was identified as a factor that stimulates the binding of methionylated initiator tRNA (Met-tRNA _i ^Met ) to the 40S ribosomal subunit, but its physiological role remains poorly defined. Recently, eIF2A was shown to be involved in unconventional translation initiation from CUG codons and in viral protein synthesis under stress conditions where eIF2 is inactivated. We determined the crystal structure of the WD-repeat domain of Schizosaccharomyces pombe eIF2A at 2.5 Å resolution. The structure adopts a novel nine-bladed β-propeller fold. In contrast to the usual β-propeller proteins, the central channel of the molecule has the narrower opening on the bottom of the protein and the wider opening on the top. Highly conserved residues are concentrated in the positively-charged top face, suggesting the importance of this face for interactions with nucleic acids or other initiation factors.     

Inheritance of the background shell color in the snails Littorina obtusata (Gastropoda,Littorinidae)

We investigated in a gastropod mollusk Littorina obtusata (L. obtusata) the inheritance of back-ground shell coloration of the shell, which arises on the basis of three pigments: purple, orange, and yellow. We found that the hypothesis on polyallelic inheritance, as in the genus Cepaea, cannot explain the inheritance of shell color in periwinkles. We demonstrated that a separate genetic system is responsible for incorporation of each pigment into the shell. The composition of these genetic systems includes at least two genes each in the case of the yellow and purple pigments. Our analysis shows that caution should be applied when extending the results obtained in the studies of the Cepaea genus to the other species of gastropods.     

The Action of 11-cis-Retinol on Cone Opsins and Intact Cone Photoreceptors

11-cis-Retinol has previously been shown in physiological experiments to promote dark adaptation and recovery of photoresponsiveness of bleached salamander red cones but not of bleached salamander red rods. The purpose of this study was to evaluate the direct interaction of 11-cis-retinol with expressed human and salamander cone opsins, and to determine by microspectrophotometry pigment formation in isolated salamander photoreceptors. We show here in a cell-free system using incorporation of radioactive guanosine 5′-3-O-(thio)triphosphate into transducin as an index of activity, that 11-cis-retinol inactivates expressed salamander cone opsins, acting an inverse agonist. Similar results were obtained with expressed human red and green opsins. 11-cis-Retinol had no significant effect on the activity of human blue cone opsin. In contrast, 11-cis-retinol activates the expressed salamander and human red rod opsins, acting as an agonist. Using microspectrophotometry of salamander cone photoreceptors before and after bleaching and following subsequent treatment with 11-cis-retinol, we show that 11-cis-retinol promotes pigment formation. Pigment was not formed in salamander red rods or green rods (containing the same opsin as blue cones) treated under the same conditions. These results demonstrate that 11-cis-retinol is not a useful substrate for rod photoreceptors although it is for cone photoreceptors. These data support the premise that rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type. These mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.11-cis-Retinol is the precursor to 11-cis-retinal, the 11-cis-aldehyde form of vitamin A and the chromophore that combines covalently with rod and cone opsin proteins to form visual pigments. 11-cis-Retinal is consumed during visual signaling, and its continual synthesis is required. Photon absorption by the visual pigments causes the isomerization of its chromophore to the all-trans configuration. This initiates two processes critical for vision: activation of the photoreceptor cell and the eventual recovery of the original photosensitivity of the cells, requiring regeneration of the visual pigments. As cones are used for bright light vision, these two processes must work more rapidly in cones than in rods and thus cones have a higher requirement of 11-cis-retinoids as suggested by Rushton (1, 2).Photoreceptor activation begins with photoisomerization of the chromophore within the visual pigment. This results in a subsequent conformational change of the protein part of the visual pigment that is able to activate its G protein transducin, which in turn activates a PDE that lowers the concentration of cGMP and closes cGMP-gated ion channels. These steps comprise the visual signal transduction cascade (see Ref. 3 for review).The visual cycle involves regeneration of the visual pigment, which ultimately deactivates the protein and accomplishes the recovery of the photosensitivity of the photoreceptor cell. Classically, this process involves both the photoreceptor cell and the retinal pigment epithelium (RPE).⁴ After photoisomerization of the chromophore and formation of the active visual pigment, all-trans-retinal is released from the opsin and reduced to all-trans-retinol, which is then transported to the RPE where it is isomerized to 11-cis-retinol through a number of steps. In the RPE, 11-cis-retinol is oxidized to the aldehyde form, which is transported back to the photoreceptor cell and can be directly used by all of the opsins to regenerate an inactive pigment ready for photoactivation. The details of this model have been extensively reviewed (4, 5). Alternatively, recent work suggests that cones have an additional source of 11-cis-retinoids from Müller cells (6–8). Like the RPE cells, Müller cells have been shown to be able to convert all-trans-retinol to 11-cis-retinol (6). Unlike in the RPE cells, 11-cis-retinol is not oxidized to 11-cis-retinal in Müller cells.Jones et al. (9) demonstrated that administration of 11-cis-retinol to bleached salamander red cones could restore photosensitivity. A logical conclusion was that red cones were able to oxidize 11-cis-retinol to the aldehyde and regenerate visual pigments although noncovalent binding of 11-cis-retinol to red cone opsins generating a light-sensitive complex could not be excluded. On the other hand, 11-cis-retinol does not restore photosensitivity to bleached salamander rod cells but appears to directly activate the cells (9, 10). The data suggested that the rods were not able to oxidize 11-cis-retinol, but that the retinol itself could activate the signal transduction cascade, and indeed we recently demonstrated that 11-cis-retinol acts as an agonist to expressed bovine rod opsin (11). Our aim here was to study the action of 11-cis-retinol on cone opsins and cone photoreceptor cells to determine the efficacy of an alternate visual cycle for cones.The photoreceptor cells used in this study are from tiger salamander, and the expressed opsins used for biochemical experiments are those from salamander and human. Photoreceptor cells are generally identified by cell morphology and the type of opsin it contains that can be further complicated by the findings that some cone cells have multiple opsins (12, 13). Recently genetic analysis has determined that opsins fall into five classes (reviewed in Refs. 14 and 15). We have studied opsins falling into four of these classes and use common color-derived names for the opsins and photoreceptor cells. The classic rod cells used for scotopic vision contain rhodopsin, the visual pigment for the rod opsin (RH1 opsin) and appeared red and thus have been designated as red rods. Some species such as salamanders have an additional rod cell whose photosensitivity is blue-shifted from that of the red rod and thus designated as green rods. In the tiger salamander, the green rods contain the identical opsin (SWS2 opsin) found in blue cones (16). The human blue cones contain an opsin from a different class (SWS1 opsin), which is homologous to the salamander UV cone opsin. The human red and green and salamander red cone opsins all belong to the same class of opsins (M/LWS opsins). Absorption properties of visual pigments are further modulated in some animals including the tiger salamander by use of 11-cis-retinal with an additional double bond (3,4-dehydro or A2 11-cis-retinal) resulting in red-shifted absorbance from pigments containing 11-cis-retinal (A1 11-cis-retinal).We show here that 11-cis-retinol is not an agonist to cone opsins and does not itself generate a light-sensitive opsin. We further show using microspectrophotometry that both red and blue salamander cone cells regenerate visual pigments from 11-cis-retinol, whereas pigments could not be regenerated with 11-cis-retinol in bleached salamander red and green rods even though the latter contains the same opsin as the salamander blue cone. Thus, rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type, and these mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.     

Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency,the PEX5 mRNA and protein levels and induction of peroxisomal deficiency

The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine^® 2000, FuGENE 6, HiPerFect^®, INTERFERin?, RiboJuice?) as well as microporation using the Neon? Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0–70 %), to the reduction of PEX5 mRNA (by 90 vs. 0–50 %) and PEX5 protein levels (by 70 vs. 0–50 %). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3′ fragment (15 %) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded to transiently knockdown PEX5 mRNA and its protein level leading to functional consequences similar as observed in peroxisome deficiencies.     

The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium The designation H. salinarium instead of the former designation H. halobium is used throughout this paper following the classification of Tindall (1992) . This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.     

Structure prediction of Bestrophin for the induced - fit docking of anthocyanins

Bestrophin, an integral membrane protein existing in basolateral region of the retina is a propitious target for drug discovery. Mutations in the Bestrophin protein cause Best Vitelliform Macular Dystrophy (BVMD) leading to retinal damages and loss of visual acuity. Owing to the lack of three dimensional structure and related structural homologs in the protein data bank, we modeled the bestrophin protein using Robetta ab initio method. Further, no treatment is available for the disease. In this situation, anthocyanins from natural sources are reported to combat retinal damages. Hence, we identified anthocyanins from Syzygium cumini fruit skin using Electrospray Ionization tandem mass spectrometry. These compounds were docked into the predicted bestrophin model to study the interactions within the active site. The results may provide a valuable insight into the structure of bestrophin and efficacy of anthocyanins in molecular docking studies.

Abbreviations

PTP - Putative transmembrane proteins, VMD - Vitelliform macular dystrophy, BVMD - Best''s vitelliform macular dystrophy, RPE - Retinal pigment epithelium, ESI-MS/MS - Electrospray Ionization Tandem Mass Spectrometry, UNIPROT - Universal Protein Resource, PSIPRED - Protein secondary structure prediction, TMH - Transmembrane Helices, SCFS - Syzygium cumini fruit skin DP - Declustering Potential IFD - Induced Fit Docking.     

Studies on the conformational state of the chromophore group (11-cis-retinal) in rhodopsin by computer molecular simulation methods

The molecular dynamics of the rhodopsin chromophore (11-cis-retinal) has been followed over a 3-ns path, whereby 3 × 10⁶ discrete conformational states of the molecule were recorded. It is shown that within a short time, 0.3–0.4 ns from the start of simulation, the retinal β-ionone ring rotates about the C6–C7 bond through ～60° relative to the initial configuration, and the whole chromophore becomes twisted. The results of ab initio quantum chemical calculations indicate that for the final conformation of the chromophore center (t = 3 ns) the rhodopsin absorption maximum is shifted by 10 nm toward longer wavelengths as compared with the initial state (t = 0). In other words, the energy of transition of such a system into the excited singlet state S1 upon photon capture will be lower than that for the molecule where the β-ionone ring of the chromophore is coplanar to its polyene chain.     

Expression inE. coli of the cloned cDNA for the major antigen of foot and mouth disease virus Asia 1 63/72

Double stranded cDNA for the foot and mouth disease virus was prepared, restricted withBamH 1 or ligated to linkers withBamH 1 sticky ends and cloned inBamH 1 site in the expression vector, pU R222. The cDNA was also cloned at thePst 1 site in the same vector by the dC/dG tailing method. They were transferred intoE. coli to give colourless colonies in the presence of the dye, X-gal. Many of them showed positive signal on hybridization with³²P-labelled viral RNA. The middleBamH1 fragment of the cDNA is known to carry the gene for the major antigen and some non-structural proteins. The clones carrying the recombinant DNA produced proteins which cross-reacted with the antibodies generated against the structural proteins of the virus in an enzyme linked immunosorbent assay, indicating that the cDNA of the major antigen is expressed in the cloned cell.