The Two-Photon Reversible Reaction of the Bistable Jumping Spider Rhodopsin-1

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore—attached to the protein through a protonated Schiff base—and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.     

RiTE database: a resource database for genus-wide rice genomics and evolutionary biology

Background

Comparative evolutionary analysis of whole genomes requires not only accurate annotation of gene space, but also proper annotation of the repetitive fraction which is often the largest component of most if not all genomes larger than 50 kb in size.

Results

Here we present the Rice TE database (RiTE-db) - a genus-wide collection of transposable elements and repeated sequences across 11 diploid species of the genus Oryza and the closely-related out-group Leersia perrieri. The database consists of more than 170,000 entries divided into three main types: (i) a classified and curated set of publicly-available repeated sequences, (ii) a set of consensus assemblies of highly-repetitive sequences obtained from genome sequencing surveys of 12 species; and (iii) a set of full-length TEs, identified and extracted from 12 whole genome assemblies.

Conclusions

This is the first report of a repeat dataset that spans the majority of repeat variability within an entire genus, and one that includes complete elements as well as unassembled repeats. The database allows sequence browsing, downloading, and similarity searches. Because of the strategy adopted, the RiTE-db opens a new path to unprecedented direct comparative studies that span the entire nuclear repeat content of 15 million years of Oryza diversity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1762-3) contains supplementary material, which is available to authorized users.     

Beta-Arrestin Functionally Regulates the Non-Bleaching Pigment Parapinopsin in Lamprey Pineal

The light response of vertebrate visual cells is achieved by light-sensing proteins such as opsin-based pigments as well as signal transduction proteins, including visual arrestin. Previous studies have indicated that the pineal pigment parapinopsin has evolutionally and physiologically important characteristics. Parapinopsin is phylogenetically related to vertebrate visual pigments. However, unlike the photoproduct of the visual pigment rhodopsin, which is unstable, dissociating from its chromophore and bleaching, the parapinopsin photoproduct is stable and does not release its chromophore. Here, we investigated arrestin, which regulates parapinopsin signaling, in the lamprey pineal organ, where parapinopsin and rhodopsin are localized to distinct photoreceptor cells. We found that beta-arrestin, which binds to stimulated G protein-coupled receptors (GPCRs) other than opsin-based pigments, was localized to parapinopsin-containing cells. This result stands in contrast to the localization of visual arrestin in rhodopsin-containing cells. Beta-arrestin bound to cultured cell membranes containing parapinopsin light-dependently and translocated to the outer segments of pineal parapinopsin-containing cells, suggesting that beta-arrestin binds to parapinopsin to arrest parapinopsin signaling. Interestingly, beta-arrestin colocalized with parapinopsin in the granules of the parapinopsin-expressing cell bodies under light illumination. Because beta-arrestin, which is a mediator of clathrin-mediated GPCR internalization, also served as a mediator of parapinopsin internalization in cultured cells, these results suggest that the granules were generated light-dependently by beta-arrestin-mediated internalization of parapinopsins from the outer segments. Therefore, our findings imply that beta-arrestin-mediated internalization is responsible for eliminating the stable photoproduct and restoring cell conditions to the original dark state. Taken together with a previous finding that the bleaching pigment evolved from a non-bleaching pigment, vertebrate visual arrestin may have evolved from a “beta-like” arrestin by losing its clathrin-binding domain and its function as an internalization mediator. Such changes would have followed the evolution of vertebrate visual pigments, which generate unstable photoproducts that independently decay by chromophore dissociation.     

Anticonvulsive effect of paeoniflorin on experimental febrile seizures in immature rats: possible application for febrile seizures in children

Febrile seizures (FS) is the most common convulsive disorder in children, but there have been no clinical and experimental studies of the possible treatment of FS with herbal medicines, which are widely used in Asian countries. Paeoniflorin (PF) is a major bioactive component of Radix Paeoniae alba, and PF-containing herbal medicines have been used for neuromuscular, neuropsychiatric, and neurodegenerative disorders. In this study, we analyzed the anticonvulsive effect of PF and Keishikashakuyaku-to (KS; a PF-containing herbal medicine) for hyperthermia-induced seizures in immature rats as a model of human FS. When immature (P5) male rats were administered PF or KS for 10 days, hyperthermia-induced seizures were significantly suppressed compared to control rats. In cultured hippocampal neurons, PF suppressed glutamate-induced elevation of intracellular Ca(2+) ([Ca(2+)](i)), glutamate receptor-mediated membrane depolarization, and glutamate-induced neuronal death. In addition, PF partially suppressed the elevation in [Ca(2+)](i) induced by activation of the metabotropic glutamate receptor 5 (mGluR5), but not that mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. However, PF did not affect production or release of γ-aminobutyric acid (GABA) in hippocampal neurons. These results suggest that PF or PF-containing herbal medicines exert anticonvulsive effects at least in part by preventing mGluR5-dependent [Ca(2+)](i) elevations. Thus, it could be a possible candidate for the treatment of FS in children.     

Structure-dependent pseudoreceptor intracellular traffic of adamantyl globotriaosyl ceramide mimics

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.     

Non‐exponential growth of Mycobacterium leprae Thai‐53 strain cultured in vitro

In this study, attempts were made to culture this bacterium in media supplemented with a variety of biological materials to determine why cultivation of Mycobacterium leprae in vitro has not this far been successful. A slight increase in the number of cells in medium supplemented with human blood plasma and an extract of nude mouse tissue as observed after more than 3 months of cultivation at 30 °C. To ascertain whether this increase was real growth, the growth was analyzed by droplet digital PCR, which showed a slow increase in the copy number of cell‐associated DNA and the release of a large amount of DNA into the culture medium from bacterial cells during cultivation. These results were supported by electron microscopic examination of M. leprae in infected mouse tissues, which showed that most of the replicated bacteria had degenerated and only a few cells survived. Based on these results, it was postulated that many of the replicated cells degenerate during M. leprae growth and that only a few cells remain to participate in the next growth stage. This means that, unlike other cultivable bacteria, the growth of M. leprae is not exponential and the number of cells therefore increase extremely slowly. Thus, accurate judging of the success of M. leprae cultivation requires observation of growth over a long period of time and careful measurement of the increase in number of viable cells.     

RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1

Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at sites of UV irradiation-induced DNA damage. In this study, we show that RAD18 is also recruited to ionizing radiation (IR)-induced sites of DNA double-strand breaks (DSBs) forming foci which are co-localized with 53BP1, NBS1, phosphorylated ATM, BRCA1 and γ-H2AX. RAD18 associates with 53BP1 and is recruited to DSB sites in a 53BP1-dependent manner specifically during G1-phase, RAD18 monoubiquitinates KBD domain of 53BP1 at lysine 1268 in vitro. A monoubiquitination-resistant 53BP1 mutant harboring a substitution at lysine 1268 is not retained efficiently at the chromatin in the vicinity of DSBs. In Rad18-null cells, retention of 53BP1 foci, efficiency of DSB repair and post-irradiation viability are impaired compared with wild-type cells. Taken together, these results suggest that RAD18 promotes 53BP1-directed DSB repair by enhancing retention of 53BP1, possibly through an interaction between RAD18 and 53BP1 and the modification of 53BP1.     

Activation by a new synthetic acyltripeptide and its analogs entrapped in liposomes of rat alveolar macrophages to the tumor cytotoxic state

Summary FK-565 (heptanoyl--d-Glu-(l-meso-a, -A₂pm (l)-d-AlaOH) is a synthetic acyltripeptide closely resembling cell wall peptidoglycan peptides of Streptomyces in structure. Alveolar macrophages (AM) lavaged from lungs of F344 rats were activated by in vitro treatment with FK-565 and its derivatives at concentrations of 1–50 g/ml medium, and the activated AM killed syngeneic mammary adenocarcinoma cells. When FK-565 and related compounds were encapsulated in multilamellar (MLV) liposomes composed of phosphatidyl-choline and phosphatidylserine, dose-response experiments showed that they were about 800 times more effective than the free compounds in activating AM. Liposome-encapsulated FK-565 and its analogs caused significant activation of AM within 4 h. These data indicated that acyltripeptide and its analogs encapsulated in liposomes are more efficient than the free compounds in rendering AM tumoricidal.     

Separation and Partial Characterization of Membranes from Prochloron sp.

Two differently colored membrane preparations were separatedfrom the prochlorophyte, Prochloron sp., by mechanical disintegrationof the cells followed by sucrose density gradient centrifugation.An orange-colored preparation, containing zeaxanthin as themajor constituent pigment, seemed to comprise the cytoplasmicmembrane. The other green-colored membrane preparation, containingß-carotene and chlorophyll a and b as major pigmentconstituents, was identified as the thylakoid membrane. Thetwo types of membranes were compared as to their absorptionspectra and buoyant densities. ¹ This work is one of the results of the 8th International Expeditionon Prochloron organized by Dr. R. A. Lewin, University of Californiaat San Diego. ⁵ Present address: Solar Energy Research Group, The Algatron,The Institute of Physical and Chemical Research (RIKEN), Wako-shi,Saitama 351, Japan. ⁶ Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received October 19, 1984; Accepted January 7, 1985)