Opening mechanism of a cyclic nucleotide-gated channel based on analysis of single channels locked in each liganded state.

Cyclic nucleotide-gated channels contain four subunits, each with a binding site for cGMP or cAMP in the cytoplasmic COOH-terminal domain. Previous studies of the kinetic mechanism of activation have been hampered by the complication that ligands are continuously binding and unbinding at each of these sites. Thus, even at the single channel level, it has been difficult to distinguish changes in behavior that arise from a channel with a fixed number of ligands bound from those that occur upon the binding and unbinding of ligands. For example, it is often assumed that complex behaviors like multiple conductance levels and bursting occur only as a consequence of changes in the number of bound ligands. We have overcome these ambiguities by covalently tethering one ligand at a time to single rod cyclic nucleotide-gated channels (Ruiz, ML., and J.W. Karpen. 1997. Nature. 389:389-392). We find that with a fixed number of ligands locked in place the channel freely moves between three conductance states and undergoes bursting behavior. Furthermore, a thorough kinetic analysis of channels locked in doubly, triply, and fully liganded states reveals more than one kinetically distinguishable state at each conductance level. Thus, even when the channel contains a fixed number of bound ligands, it can assume at least nine distinct states. Such complex behavior is inconsistent with simple concerted or sequential allosteric models. The data at each level of liganding can be successfully described by the same connected state model (with different rate constants), suggesting that the channel undergoes the same set of conformational changes regardless of the number of bound ligands. A general allosteric model, which postulates one conformational change per subunit in both the absence and presence of ligand, comes close to providing enough kinetically distinct states. We propose an extension of this model, in which more than one conformational change per subunit can occur during the process of channel activation.     

Polyplex‐mediated gene transfer into human retinal pigment epithelial cells in vitro

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age‐related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome‐mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex‐mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20\% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex‐mediated gene transfer, but stable integration does occur at low frequencies with and without selection. J. Cell. Biochem. 76:153–160, 1999. © 1999 Wiley‐Liss, Inc.     

Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: A study of retinal regeneration in a novel animal model

Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three‐dimensional (3‐D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin‐positive cells nor 3‐D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 739–756, 2014     

Stem‐cell‐based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age‐related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem‐cell‐derived retinal pigment epithelial cells for AMD treatment and stem cell‐derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.     

Bacterial periphyton of the Vltava River

Bacterial periphyton formed during 48 hours was studied by glass slide method and direct counting in Vltava River in Praha, Czechoslovakia. At water temperatures 8–11°C the numbers of rods ranged between 24,000 and 336,000 per 1 cm² and those of cocci between 30,000 and 228,000. The relation rods: cocci ranged between 0,9 and 2,4 with an average value of 1,7, whereas in a fishpond this average was 0,5. Among the periphyton 81,3% bacterial cells were active. The rods: cocci relation seems to be a good indicator of water pollution by organic matter, but numbers distinguishing the individual saprobic levels cannot be given yet.

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Zusammenfassung Es wurde der bakterielle Bewuchs von Glassplatten im Vltava-Fluss in Praha untersucht. Zweitägige Exposition erwies sich als am meisten geeignet, um den Bewuchs mikroskopisch durch direkte Zählung quantitativ zu erfassen. In Abhängigkeit von der Tiefe, Wassertemperatur, Verunreinigungsgrad, Sonnenstrahlung u.a. entwickelte sich der Bewuchs quantitativ unterschiedlich und zeigte auch eine unterschiedliche Relation von Stäbchen zu Kokken. Im beta-mesosaproben Bereich wurden im Herbst (Wassertemperaturen 8–11 °C) 24.000 bis 336.000 Stäbchen und 30.000 bis 228.000 Kokken per 1 cm² gefunden, mit den Mittelwerten 163.100 Stäbchen und 104.100 Kokken. Die Vergleichswerte von einem Fischteich in Motol lagen für Stäbchen im gleichen Bereich, während die Kokken, bis 254.000 erreichten.Die Relation Stäbchen: Kokken variierte im Vltava-Fluss zwischen 0,9 (nur einmal) und 2,4 mit dem Mittelwert 1,7, im Fischteich zwischen 0,4 ufnd 0,7 mit dem Mittelwert 0,5. Es hat sich erwiesen, dass diese Relation brauchbare Angaben über die organische Verunreinigung gibt. Leider liegen bisher zu wenige Ergebnisse vor, um eine Skale gegenüber den Saprobitätsstufen zu errichten.Unter den Bewuchsbakterien gab es in der Vltava durchschnittlich 81,3% aktive Zellen, wie durch Fluoreszenzanalyse festgestellt wurde.Fast alle Daten indizieren eine schwächere Verunreinigung des Wassers am linken Ufer als am rechten.

     

A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc.     

Deciphering the mechanisms of intestinal imino (and amino) acid transport: The redemption of SLC36A1

The absorption of zwitterionic imino and amino acids, and related drugs, is an essential function of the small intestinal epithelium. This review focuses on the physiological roles of transporters recently identified at the molecular level, in particular SLC36A1, by identifying how they relate to the classical epithelial imino and amino acid transporters characterised in mammalian small intestine in the 1960s-1990s. SLC36A1 transports a number of d- and l-imino and amino acids, β- and γ-amino acids and orally-active neuromodulatory and antibacterial agents. SLC36A1 (or PAT1) functions as a proton-coupled imino and amino acid symporter in cooperation with the Na⁺/H⁺ exchanger NHE3 (SLC9A3) to produce the imino acid carrier identified in rat small intestine in the 1960s but subsequently ignored because of confusion with the IMINO transporter. However, it is the sodium/imino and amino acid cotransporter SLC6A20 which corresponds to the betaine carrier (identified in hamster, 1960s) and IMINO transporter (identified in rabbit and guinea pig, 1980s). This review summarises evidence for expression of SLC36A1 and SLC6A20 in human small intestine, highlights the differences in functional characteristics of the imino acid carrier and IMINO transporter, and explains the confusion surrounding these two distinct transport systems.     

Phospholipid meets all-trans-retinal: the making of RPE bisretinoids

The lipid phase of the photoreceptor outer segment membrane is essential to the photon capturing and signaling functions of rhodopsin. Rearrangement of phospholipids in the bilayer accompanies the formation of the active intermediates of rhodopsin following photon absorption. Furthermore, evidence for the formation of a condensation product between the photolyzed chromophore all-trans-retinal and phosphatidylethanolamine indicates that phospholipid may also participate in the movement of the retinoid in the membrane. The downside of these interactions is the formation of bisretinoid-phosphatidylethanolamine compounds that accumulate in retinal pigment epithelial cells with age and that are particularly abundant in some retinal disorders. The propensity of these compounds to negatively impact on the cells has been linked to the pathogenesis of some retinal disorders including juvenile onset recessive Stargardt disease and age-related macular degeneration.