Segment Membranes Research Articles

Chemistry and biology of the initial steps in vision: the Friedenwald lecture.

Visual transduction is the process in the eye whereby absorption of light in the retina is translated into electrical signals that ultimately reach the brain. The first challenge presented by visual transduction is to understand its molecular basis. We know that maintenance of vision is a continuous process requiring the activation and subsequent restoration of a vitamin A-derived chromophore through a series of chemical reactions catalyzed by enzymes in the retina and retinal pigment epithelium (RPE). Diverse biochemical approaches that identified key proteins and reactions were essential to achieve a mechanistic understanding of these visual processes. The three-dimensional arrangements of these enzymes' polypeptide chains provide invaluable insights into their mechanisms of action. A wealth of information has already been obtained by solving high-resolution crystal structures of both rhodopsin and the retinoid isomerase from pigment RPE (RPE65). Rhodopsin, which is activated by photoisomerization of its 11-cis-retinylidene chromophore, is a prototypical member of a large family of membrane-bound proteins called G protein-coupled receptors (GPCRs). RPE65 is a retinoid isomerase critical for regeneration of the chromophore. Electron microscopy (EM) and atomic force microscopy have provided insights into how certain proteins are assembled to form much larger structures such as rod photoreceptor cell outer segment membranes. A second challenge of visual transduction is to use this knowledge to devise therapeutic approaches that can prevent or reverse conditions leading to blindness. Imaging modalities like optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) applied to appropriate animal models as well as human retinal imaging have been employed to characterize blinding diseases, monitor their progression, and evaluate the success of therapeutic agents. Lately two-photon (2-PO) imaging, together with biochemical assays, are revealing functional aspects of vision at a new molecular level. These multidisciplinary approaches combined with suitable animal models and inbred mutant species can be especially helpful in translating provocative cell and tissue culture findings into therapeutic options for further development in animals and eventually in humans. A host of different approaches and techniques is required for substantial progress in understanding fundamental properties of the visual system.

Simultaneous Purification of Limonin, Nomilin and Isoobacunoic Acid from Pomelo Fruit (Citrus grandis) Segment Membrane

A method was established for purification of limonin, nomilin, and isoobacunoic acid simultaneously from segment membranes of pomelo (Citrus Grandis). This method includes 3 steps, removing most impurities by macroporous resin HZ-816, isolating limonin by High Speed Counter Current Chromatography (HSCCC), and isolating nomilin and isoobacunoic acid by semi-preparative HPLC. Naringin was partially purified as a by-product of this process using Sephadex LH-20. All limonoids purified through this method reached 95% purity. The purified limonin, nomilin and isoobacunoic acid were identified according to the retention time of the standard substances using HPLC and characteristic fragment ions of LC-MS/MS. A method of getting health care products limonoids from fruit processing by-products segments membrane of pomelo.

Chemopreventive effect of flavonoids from Ougan (Citrus reticulata cv. Suavissima) fruit against cancer cell proliferation and migration

Citrus reticulata cv. Suavissima (Ougan) is a rich source of flavonoids, and its peel has been employed as a traditional Chinese medicine for a long time. Among the different fruit parts, namely flavedo, albedo, segment membrane and juice sacs, flavedo showed significant inhibitory effect on three cancer cell lines (HepG2, HL-60 and MDA-MB-231). Three flavanone glycosides, naringin, hesperidin, neohesperidin as well as four polymethoxylated flavones (PMFs), sinensetin, nobiletin, tangeretin and 5-demethylnobiletin were separated and purified from Ougan flavedo and identified by HPLC-DAD and LC-MS. Cytotoxicity assay showed that two flavanone aglycones and four PMFs exhibited significant antiproliferative activity on the three cancer cells. Structure–activity relationship was observed for the Ougan flavonoid-mediated antiproliferative activity. Wound healing assay showed that nobiletin and tangeretin exerted inhibitory effects on SKOV3 cell migration as well. These results indicate that Ougan flavonoids may have preventive or therapeutic potential for inhibiting cancer cell proliferation and migration.

R9AP targeting to rod outer segments is independent of rhodopsin and is guided by the SNARE homology domain.

In vertebrate photoreceptor cells, rapid recovery from light excitation is dependent on the RGS9⋅Gβ5 GTPase-activating complex located in the light-sensitive outer segment organelle. RGS9⋅Gβ5 is tethered to the outer segment membranes by its membrane anchor, R9AP. Recent studies indicated that RGS9⋅Gβ5 possesses targeting information that excludes it from the outer segment and that this information is overridden by association with R9AP, which allows outer segment targeting of the entire complex. It was also proposed that R9AP itself does not contain specific targeting information and instead is delivered to the outer segment in the same post-Golgi vesicles as rhodopsin, because they are the most abundant transport vesicles in photoreceptor cells. In this study, we revisited this concept by analyzing R9AP targeting in rods of wild-type and rhodopsin-knockout mice. We found that the R9AP targeting mechanism does not require the presence of rhodopsin and further demonstrated that R9AP is actively targeted in rods by its SNARE homology domain.

A Transient Interaction between the Phosphate Binding Loop and Switch I Contributes to the Allosteric Network between Receptor and Nucleotide in Gαi1

Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit.

ROS-GC interlocked Ca(2+)-sensor S100B protein signaling in cone photoreceptors: review.

Photoreceptor rod outer segment membrane guanylate cyclase (ROS-GC) is central to visual transduction; it generates cyclic GMP, the second messenger of the photon signal. Photoexcited rhodopsin initiates a biochemical cascade that leads to a drop in the intracellular level of cyclic GMP and closure of cyclic nucleotide gated ion channels. Recovery of the photoresponse requires resynthesis of cyclic GMP, typically by a pair of ROS-GCs, 1 and 2. In rods, ROS-GCs exist as complexes with guanylate cyclase activating proteins (GCAPs), which are Ca2+-sensing elements. There is a light-induced fall in intracellular Ca2+. As Ca2+ dissociates from GCAPs in the 20–200 nM range, ROS-GC activity rises to quicken the photoresponse recovery. GCAPs then progressively turn down ROS-GC activity as Ca2+ and cyclic GMP levels return to baseline. To date, GCAPs mediate the only known mechanism of ROS-GC regulation in the photoreceptors. However, in mammalian cone outer segments, cone synapses and ON bipolar cells, another Ca2+ sensor protein, S100B, complexes with ROS-GC1 and senses the Ca2+ signal with a K1/2 of 400 nM. Unlike GCAPs, S100B stimulates ROS-GC activity when Ca2+ is bound. Thus, the ROS-GC system in cones functions as a Ca2+ bimodal switch; with rising intracellular Ca2+, its activity is first turned down by GCAPs and then turned up by S100B. This presentation provides a historical perspective on the role of S100B in the photoreceptors, offers a pictorial model for the “bimodal” operation of the ROS-GC switch and projects future tasks that are needed to understand its operation. Some accounts of this review have been adopted from the original publications of these authors.

Distinct functions for IFT140 and IFT20 in opsin transport

In the vertebrate retina, light is detected by the outer segments of photoreceptor rods and cones, which are highly modified cilia. Like other cilia, outer segments have no protein synthetic capacity and depend on proteins made in the cell body for their formation and maintenance. The mechanism of transport into the outer segment is not fully understood but intraflagellar transport (IFT) is thought to be a major mechanism for moving protein from the cell body into the cilium. In the case of photoreceptor cells, the high density of receptors and the disk turnover that occurs daily necessitates much higher rates of transport than would be required in other cilia. In this work, we show that the IFT complex A protein IFT140 is required for development and maintenance of outer segments. In earlier work we found that acute deletion of Ift20 caused opsin to accumulate at the Golgi complex. In this work, we find that acute deletion of Ift140 does not cause opsin to accumulate at the Golgi complex but rather it accumulates in the plasma membrane of the inner segments. This work is a strong support of a model of opsin transport where IFT20 is involved in the movement from the Golgi complex to the base of the cilium. Then, once at the base, the opsin is carried through the connecting cilium by an IFT complex that includes IFT140. © 2014 Wiley Periodicals, Inc.

Activities of enzymes directly related with sucrose and citric acid metabolism in citrus fruit in response to soil plastic film mulch

Soil plastic film mulch is commonly employed in citrus production regions of East Asia to improve fruit quality. In the present study, Ponkan tangerine (Citrus reticulata Blanco) was mulched under the tree canopy with silver-black reflective film during fruit development. At about 12 days after mulching, total soluble sugar and citric acid contents in the segment membrane and/or juice sacs of fruit from mulched trees increased significantly relative to the control. In the segment membrane, the activities of acid invertase (AI) and sucrose synthase (SS; cleavage direction) increased significantly following mulch treatment. However, the activities of other enzymes, including neutral invertase, SS (synthetic direction) and sucrose phosphate synthase did not respond significantly under mulch treatment. In the juice sacs, SS activity (cleavage direction) from mulched treatments was significantly lower than that from control trees while SS activity (synthetic direction) from mulched trees was significantly higher than that from control trees. Moreover, the activities of cytoplasm aconitase (cyt-Aco) and isocitrate dehydrogenase (cyt-IDH) were significantly lower than those in the control fruits after 36 days of mulching. In conclusion, the activities of SS (synthetic direction) and AI were significantly enhanced while those of cyt-Aco and cyt-IDH were significantly reduced by soil plastic film mulch. A schematic model is present indicating the possible important roles that these key enzymes play in sugar and acid accumulation in citrus fruits under soil plastic film mulch.

Novel endogenous glycan therapy for retinal diseases: safety, in vitro stability, ocular pharmacokinetic modeling, and biodistribution.

Asialo, tri-antennary oligosaccharide (NA3 glycan) is an endogenous compound, which supports proper folding of outer segment membranes, promotes normal ultrastructure, and maintains protein expression patterns of photoreceptors and Müller cells in the absence of retinal pigment epithelium support. It is a potential new therapeutic for atrophic age-related macular degeneration (AMD) and other retinal degenerative disorders. Herein, we evaluate the safety, in vitro stability, ocular pharmacokinetics and biodistribution of NA3. NA3 was injected into the vitreous of New Zealand white rabbits at two concentrations viz. 1 nM (minimum effective concentration (MEC)) and 100 nM (100XMEC) at three time points. Safety was evaluated using routine clinical and laboratory tests. Ocular pharmacokinetics and biodistribution of [(3)H]NA3 were estimated using scintillation counting in various parts of the eye, multiple peripheral organs, and plasma. Pharmacokinetic parameters were estimated by non-compartmental modeling. A 2-aminobenzamide labeling and hydrophilic interaction liquid interaction chromatography were used to assess plasma and vitreous stability. NA3 was well tolerated by the eye. The concentration of NA3 in eye tissues was in the order: vitreous > retina > sclera/choroid > aqueous humor > cornea > lens. Area under the curve (0 to infinity) (AUC∞) was the highest in the vitreous thereby providing a positive concentration gradient for NA3 to reach the retina. Half-lives in critical eye tissues ranged between 40 and 60 h. NA3 concentrations were negligible in peripheral organs. Radioactivity from [(3)H]NA3 was excreted via urine and feces. NA3 was stable at 37°C in vitreous over a minimum of 6 days, while it degraded rapidly in plasma. Collectively, these results document that NA3 shows a good safety profile and favorable ocular pharmacokinetics.

Insights into the mechanisms of macular degeneration associated with the R172W mutation in RDS

Mutations in the photoreceptor tetraspanin gene peripherin-2/retinal degeneration slow (PRPH2/RDS) cause both rod- and cone-dominant diseases. While rod-dominant diseases, such as autosomal dominant retinitis pigmentosa, are thought to arise due to haploinsufficiency caused by loss-of-function mutations, the mechanisms underlying PRPH2-associated cone-dominant diseases are unclear. Here we took advantage of a transgenic mouse line expressing an RDS mutant (R172W) known to cause macular degeneration (MD) in humans. To facilitate the study of cones in the heavily rod-dominant mouse retina, R172W mice were bred onto an Nrl(-/-) background (in which developing rods adopt a cone-like fate). In this model the R172W protein and the key RDS-binding partner, rod outer segment (OS) membrane protein 1 (ROM-1), were properly expressed and trafficked to cone OSs. However, the expression of R172W led to dominant defects in cone structure and function with equal effects on S- and M-cones. Furthermore, the expression of R172W in cones induced subtle alterations in RDS/ROM-1 complex assembly, specifically resulting in the formation of abnormal, large molecular weight ROM-1 complexes. Fundus imaging demonstrated that R172W mice developed severe clinical signs of disease nearly identical to those seen in human MD patients, including retinal degeneration, retinal pigment epithlium (RPE) defects and loss of the choriocapillaris. Collectively, these data identify a primary disease-causing molecular defect in cone cells and suggest that RDS-associated disease in patients may be a result of this defect coupled with secondary sequellae involving RPE and choriocapillaris cell loss.

Phytochemical Composition, Antioxidant and Antibacterial Properties of Pummelo (<i>Citrus maxima</i> (Burm.)) Merr. against <i>Escherichia coli</i> and <i>Salmonella typhimurium</i>

The antioxidant and antibacterial activities of the phytochemical constituents of the pericarp, mesocarp and segment membrane crude ethanolic extracts of Pummelo (Citrus maxima (Burm.)) fruit were were tested against Escherichia coli and Salmonella typhimurium. Preliminary phytochemical test revealed the presence of phenols, tannins, saponins expressed as catechine quivalent (CE)/100ml and flavonoid expressed as gallic acid equivalent (GAE)/100ml. The order of which was as follows pericarp > segment membrane > mesocarp. The strongest antioxidant activity was obtained by the pericarp extract (29.64 expressed as % lipid peroxidation). The differences in the measured amount of phytochemicals and antioxidant activity among the three sample extracts were found to be significant. In terms of antimicrobial activity, the pericarp, mesocarp and segment membrane extracts generated zone of inhibitions measuring 17.10, 18.00 and 17.03 mm for S. typhimurium, respectively at 100% concentration. E. coli was noted to be inactive in all three sample extracts at 100% concentration. The capacity of E. coli to counteract the inhibitory effect of the phytochemicals contained in the pummelo extracts may be attributed to its rough corrugated cell wall and thick periplasmic space as opposed to the smooth curved and barely seen periplasmic space of S. typhimurium. However, no significant correlation was detected among the phytochemical content, antioxidant and in vitro antimicrobial activities of the sample extracts against S. typhimurium.

The Distributions and Orientations of Retinoids in Retinal Membranes Studied with All-Atom Molecular Dynamics Simulations

Understanding the transport of retinoids in lipid bilayers is essential to describe the binding to photoreceptors or retinoid-binding proteins involved in the visual cycle. Utilizing all-atom molecular simulations, we demonstrate how 11-cis retinal, all-trans retinal and all-trans retinol distribute and orient in lipid bilayers with different levels of unsaturation, head group and the presence of cholesterol, characteristic of retinal membranes. We have found that all-trans retinol is more depleted in the membrane core (with a substantially lower density relative to retinal) and less flexible in its orientation (with a more parallel alignment to the membrane normal) than its retinal counterpart. We shall discuss how these results pertain to the vision cycle. In particular, the restricted distribution of retinol may assist the removal of all-trans retinol from the rod outer segment(ROS) membranes by facilitating its binding to certain retinoid-binding proteins. On the other hand, the higher accessibility of the membrane core to all-trans retinal and its flexible orientation may assist its release from bleached photoreceptors. Furthermore, we demonstrate that the presence of cholesterol increases the concentration of 11-cis retinal in the membrane core and will explain how this is consistent with the roles of plasma and disk membranes in the retinoid cycle.

Effect of UV Crosslinking on Transport Properties of CO2 and N2 Through Poly(Imide-siloxane) Segmented Copolymer

Crosslinkable segmented random copolymers are made by synthesizing a polyimide in the presence of bisaminopropyl dimethylsiloxane (PDMS) oligomers. The polyimide segments of these materials are composed of two different dianhydrides, 2,2’-bis-(3,4-dicarboxyphenyl) hexafluoropropane dianhydride (6FDA), and 3,3’,4,4’- benzophenone tetracarboxylic dianhydride (BTDA) with the base diamine, 2,4,6-trimethyl-1,3-phenylenediamine (DAM). Upon irradiation with UV light, the ketone group in BTDA can form radicals that crosslink with adjacent benzylic methyl groups on DAM. Thermal analysis reveals that siloxane-containing polyimide copolymer films are phase segregated, composed of a polyimide-rich continuous phase and dispersed PDMS phase. CO2 and N2 gas permeabilities of uncrosslinked and crosslinked samples were measured to investigate the effect of UV crosslinking on permeability and permselectivity. Incorporation of PDMS did not significantly change the transport properties of the membranes. Densification occurred as a result of UV crosslinking, which enhanced the size-sieving capability of the poly(imide-siloxane) segmented copolymer membrane.

Supramolecular Organization of Rod Outer Segment Membrane: New Rhodopsin Dimer Interface and Insights from the β2Ar-Gs Complex

How the components of the G protein-coupled receptor (GPCR) “signalosome” assemble and function in the membrane bilayer is not known. Nevertheless the highly symmetrical organization of visual receptors (rhodopsin, rho) in rows-of-dimers suggested by AFM images of the rod cell disk membrane has a simple and direct consequence: the exposure of one unique surface of the receptor to the lipid phase of the membrane. This organization defines the encounter complex between rho and the G protein (transducin, Gt), and the orientation of rho in the dimer is therefore determinant. We have previously identified both computationally[1] and experimentally[2] a stable rho dimer involving TM1/TM2 and H8. Here we describe the investigation of alternative receptor binding modes leading to the identification of a new interface involving TM5/TM6. PMF analysis of this interface using the MARTINI coarse grain model indicates a similar order of stability as the TM1/TM2/H8 interface. However it involves a significant conformational change of a loop as compared to the ground state structure we originally used (PDB:1U19). This finding underlines the importance of loop flexibility in membrane protein interactions. Interestingly, when dimers of rho based on this new interface are arranged in rows-of-dimers, Gt placed on rho following the arrangement recently described for Gs on the β2AR correctly embed the lipid anchors of Gtα and Gtγ in the lipid phase. We also discuss the pros and cons of the different models of rho dimers for the visual phototransduction machinery, in the context of the rows-of-dimers arrangement, the functional implications of such supramolecular organization, the binding of Gt to rho and the structural, biochemical and physiological data available and the potential relevance for other GPCRs.[1] Periole, et al.(2012)JAmChemSoc134,10959-10965.[2] Knepp, et al.(2012)Biochemistry51,1819-1821.

An Inducible Expression System to Measure Rhodopsin Transport in Transgenic Xenopus Rod Outer Segments

We developed an inducible transgene expression system in Xenopus rod photoreceptors. Using a transgene containing mCherry fused to the carboxyl terminus of rhodopsin (Rho-mCherry), we characterized the displacement of rhodopsin (Rho) from the base to the tip of rod outer segment (OS) membranes. Quantitative confocal imaging of live rods showed very tight regulation of Rho-mCherry expression, with undetectable expression in the absence of dexamethasone (Dex) and an average of 16.5 µM of Rho-mCherry peak concentration after induction for several days (equivalent to >150-fold increase). Using repetitive inductions, we found the axial rate of disk displacement to be 1.0 µm/day for tadpoles at 20 °C in a 12 h dark /12 h light lighting cycle. The average distance to peak following Dex addition was 3.2 µm, which is equivalent to ~3 days. Rods treated for longer times showed more variable expression patterns, with most showing a reduction in Rho-mCherry concentration after 3 days. Using a simple model, we find that stochastic variation in transgene expression can account for the shape of the induction response.

Timing Is Everything: GTPase Regulation in Phototransduction

As the molecular mechanisms of vertebrate phototransduction became increasingly clear in the 1980s, a persistent problem was the discrepancy between the slow GTP hydrolysis catalyzed by the phototransduction G protein, transducin, and the much more rapid physiological recovery of photoreceptor cells from light stimuli. Beginning with a report published in 1989, a series of studies revealed that transducin GTPase activity could approach the rate needed to explain physiological recovery kinetics in the presence of one or more factors present in rod outer segment membranes. One by one, these factors were identified, beginning with PDEγ, the inhibitory subunit of the cGMP phosphodiesterase activated by transducin. There followed the discovery of the crucial role played by the regulator of G protein signaling, RGS9, a member of a ubiquitous family of GTPase-accelerating proteins, or GAPs, for heterotrimeric G proteins. Soon after, the G protein β isoform Gβ5 was identified as an obligate partner subunit, followed by the discovery or R9AP, a transmembrane protein that anchors the RGS9 GAP complex to the disk membrane, and is essential for the localization, stability, and activity of this complex in vivo. The physiological importance of all of the members of this complex was made clear first by knockout mouse models, and then by the discovery of a human visual defect, bradyopsia, caused by an inherited deficiency in one of the GAP components. Further insights have been gained by high-resolution crystal structures of subcomplexes, and by extensive mechanistic studies both in vitro and in animal models.

Functional asymmetry within the Sec61p translocon

The Sec61 translocon forms a pore to translocate polypeptide sequences across the membrane and offers a lateral gate for membrane integration of hydrophobic (H) segments. A central constriction of six apolar residues has been shown to form a seal, but also to determine the hydrophobicity threshold for membrane integration: Mutation of these residues in yeast Sec61p to glycines, serines, aspartates, or lysines lowered the hydrophobicity required for integration; mutation to alanines increased it. Whereas four leucines distributed in an oligo-alanine H segment were sufficient for 50% integration, we now find four leucines in the N-terminal half of the H segment to produce significantly more integration than in the C-terminal half, suggesting functional asymmetry within the translocon. Scanning a cluster of three leucines through an oligo-alanine H segment showed high integration levels, except around the position matching that of the hydrophobic constriction in the pore where integration was strongly reduced. Both asymmetry and the position effect of H-segment integration disappeared upon mutation of the constriction residues to glycines or serines, demonstrating that hydrophobicity at this position within the translocon is responsible for the phenomenon. Asymmetry was largely retained, however, when constriction residues were replaced by alanines. These results reflect on the integration mechanism of transmembrane domains and show that membrane insertion of H segments strongly depends not only on their intrinsic hydrophobicity but also on the local conditions in the translocon interior. Thus, the contribution of hydrophobic residues in the H segment is not simply additive and displays cooperativeness depending on their relative position.

Content changes of bitter compounds in ‘Guoqing No.1’ Satsuma mandarin (Citrus unshiu Marc.) during fruit development of consecutive 3 seasons

The main bitter compounds (nomilin, limonin and naringin) in the fruit tissues of ‘Guoqing No.1’ Satsuma mandarin (Citrus unshiu Marc.) were determined throughout the fruit development of 3 consecutive growing seasons. Although fluctuating largely at the corresponding developing stages of the 3years, the contents of these compounds in fruit tissues mostly displayed a declining trend, which implied that the rhythm of the metabolism of these bitter compounds was not consistent among years and was largely growing season dependent. Regarding their distribution, fruit flavedo might be a weak sink that contained the lowest level of naringin, while the segment membrane accumulated large amount of limonin and nomilin, which indicated a possible tissue bias pattern for biosynthesis or accumulation of those compounds. Partial correlation coefficient analysis revealed a synergistic accumulation of naringin and the two limonoid aglycones in fruit tissues during fruit development, indicating an integrated metabolism of flavonoids and limonoids.

Deletion of the Basigin gene results in reduced mitochondria in the neural retina

Basigin-null mice are characterized as blind from the time of eye opening, with degeneration of the retina beginning at 8weeks of age, and progressing until the entire photoreceptor cell layer is destroyed. It is likely that a metabolic deficiency underlies the blindness and degeneration phenotypes, as it has been determined that Basigin-null mice do not express the transporter protein monocarboxylate transporter one on the membrane of photoreceptor cells and inner segments, nor Müller cells of the neural retina, as is observed in normal mice. The purpose of the present study was to assess the health of mitochondria in normal and Basigin-null mice, specifically to determine if mitochondria within the Basigin-null mouse neural retina are metabolically active. This was achieved via a measurement of cytochrome C concentration and the expression of autophagy-specific proteins via ELISA analyses. Additionally, Mitotracker dyes were used to assess the number and relative activity of mitochondria. It was determined that cytochrome C concentrations and expression of autophagy-specific proteins were not increased in Basigin-null animals, as compared to control animals. Also, while Basigin-null mice do have metabolically active mitochondria, the amount of mitochondria was greatly reduced, when compared to control animals. The results suggest that a reduction in mitochondria is a result, rather than the cause, of the metabolic deficiency observed in Basigin-null mice, and likely occurs because of reduced metabolic activity in the absence of MCT1 expression.

Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F, Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.