Mice deficient in the Vici syndrome gene Epg5 exhibit features of retinitis pigmentosa

Autophagy helps to maintain cellular homeostasis by removing misfolded proteins and damaged organelles, and generally acts as a cytoprotective mechanism for neuronal survival. Here we showed that mice deficient in the Vici syndrome gene Epg5, which is required for autophagosome maturation, show accumulation of ubiquitin-positive inclusions and SQSTM1 aggregates in various retinal cell types. In epg5^?/? retinas, photoreceptor function is greatly impaired, and degenerative features including progressively reduced numbers of photoreceptor cells and increased numbers of apoptotic cells in the outer nuclear layer are observed, while the morphology of other parts of the retina is not severely affected. Downstream targets of the unfolded protein response (UPR), including the death inducer DDIT3/CHOP, and also levels of cleaved CASP3 (caspase 3), are elevated in epg5^?/? retinas. Thus, apoptotic photoreceptor cell death in epg5^?/? retinas may result from the elevated UPR. Our results reveal that Epg5-deficient mice recapitulate key characteristics of retinitis pigmentosa and thus may provide a valuable model for investigating the molecular mechanism of photoreceptor degeneration.     

Putative ClC-2 Chloride Channel Mediates Inward Rectification in <Emphasis Type="Italic">Drosophila </Emphasis>Retinal Photoreceptors

We report that Drosophila retinal photoreceptors express inwardly rectifying chloride channels that seem to be orthologous to mammalian ClC-2 inward rectifier channels. We measured inwardly rectifying Cl⁻ currents in photoreceptor plasma membranes: Hyperpolarization under whole-cell tight-seal voltage clamp induced inward Cl⁻ currents; and hyperpolarization of voltage-clamped inside-out patches excised from plasma membrane induced Cl⁻ currents that have a unitary channel conductance of ∼3.7 pS. The channel was inhibited by 1 mM Zn²⁺ and by 1 mM 9-anthracene, but was insensitive to DIDS. Its anion permeability sequence is Cl⁻ = SCN⁻> Br⁻>> I⁻, characteristic of ClC-2 channels. Exogenous polyunsaturated fatty acid, linolenic acid, enhanced or activated the inward rectifier Cl⁻ currents in both whole-cell and excised patch-clamp recordings. Using RT-PCR, we found expression in Drosophila retina of a ClC-2 gene orthologous to mammalian ClC-2 channels. Antibodies to rat ClC-2 channels labeled Drosophila photoreceptor plasma membranes and synaptic regions. Our results provide evidence that the inward rectification in Drosophila retinal photoreceptors is mediated by ClC-2-like channels in the non-transducing (extra-rhabdomeral) plasma membrane, and that this inward rectification can be modulated by polyunsaturated fatty acid. G. Ugarte and R. Delgado contributed equally to this work.     

Glycine input induces the synaptic facilitation in salamander rod photoreceptors

Glycinergic synapses in photoreceptors are made by centrifugal feedback neurons in the network, but the function of the synapses is largely unknown. Here we report that glycinergic input enhances photoreceptor synapses in amphibian retinas. Using specific antibodies against a glycine transporter (GlyT2) and glycine receptor β subunit, we identified the morphology of glycinergic input in photoreceptor terminals. Electrophysiological recordings indicated that 10 μM glycine depolarized rods and activated voltage-gated Ca²⁺ channels in the neurons. The effects facilitated glutamate vesicle release in photoreceptors, meanwhile increased the spontaneous excitatory postsynaptic currents in Off-bipolar cells. Endogenous glycine feedback also enhanced glutamate transmission in photoreceptors. Additionally, inhibition of a Cl⁻ uptake transporter NKCC1 with bumetanid effectively eliminated glycine-evoked a weak depolarization in rods, suggesting that NKCC1 maintains a high Cl⁻ level in rods, which causes to depolarize in responding to glycine input. This study reveals a new function of glycine in retinal synaptic transmission.     

A phospholipase inhibitor, manoalide, and a G-protein activator, Mas-7, both affect the turnover of phototransductive membranes by crab retinas in darkness With a model for the regulation of rhabdomeral membrane turnover

(1) In vitro retinas of a crab, Leptograpsus, were treated with a phospholipase inhibitor, manoalide, or a G-protein activator, Mas-7. Both drugs address early stages of the phototransduction cascade. (2) Manoalide inhibited the light-dependent reduction of rhabdoms during the `day' phase of the light cycle, but did not induce rhabdom overgrowth. Following a period of darkness manoalide failed to affect the diminution of illuminated rhabdoms. (3) The diminution of rhabdoms that follows photoreceptor depolarisation induced by 100 mmol · l<sup>−1</sup> K<sup>+</sup> in darkness was not affected by 2␣μmol · l<sup>−1</sup> manoalide. (4) When retinas in the `night' phase were treated with Mas-7 in darkness, rhabdom diameters were augmented, concurrently with endocytosis of photoreceptor plasma membranes. (5) The results of combining manoalide and Mas-7 with actinomycin D, U-57908 or okadaic acid, drugs used in previous studies to manipulate steps notionally lower in the transduction cascade, lead to a hypothetical model for the regulation of phototransductive membrane turnover by arthropods. Accepted: 3 October 1996     

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D₄ receptor (D₄R) activation promotes light adaptation and suppresses the light‐sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light‐ and D₄R‐signaling pathways converge on the type 1 Ca²⁺/calmodulin‐stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D₄R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D₄R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D₄R, cyclic AMP levels in the dark‐adapted retina are significantly lower compared to wild‐type retina and are unresponsive to light. These changes in Drd4?/? mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity in retinal membranes compared with wild‐type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild‐type mice treated chronically with a D₄R antagonist, L‐745870. Thus, activation of D₄R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross‐talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.     

Pre-induced adult human peripheral blood mononuclear cells migrate widely into the degenerative retinas of rd1 mice

Background aimsRecent advances in stem cell research have raised the possibility of stem cells repairing or replacing retinal photoreceptor cells that are either dysfunctional or lost in many retinal diseases. Various types of stem cells have been used to replace retinal photoreceptor cells. Recently, peripheral blood stem cells, a small proportion of pluripotent stem cells, have been reported to mainly exist in the peripheral blood mononuclear cells (PBMCs).MethodsIn this study, the effects of pre-induced adult human PBMCs (hPBMCs) on the degenerative retinas of rd1 mice were investigated. Freshly isolated adult hPBMCs were pre-induced with the use of the conditioned medium of rat retinas for 4 days and were then labeled with chloromethyl-benzamidodialkylcarbocyanine (CM-DiI) and then transplanted into the subretinal space of the right eye of rd1 mice through a trans-scleral approach. The right eyes were collected 30 days after transplantation. The survival and migration of the transplanted cells in host retinas were investigated by whole-mount retinas, retinal frozen sections and immunofluorescent staining.ResultsAfter subretinal transplantation, pre-induced hPBMCs were able to survive and widely migrate into the retinas of rd1 mice. A few CM-DiI–labeled cells migrated into the inner nuclear layer and the retinal ganglion cell layer. Some transplanted cells in the subretinal space of rd1 host mice expressed the human photoreceptor–specific marker rhodopsin.ConclusionsThis study suggests that pre-induced hPBMCs may be a potential cell source of cell replacement therapy for retinal degenerative diseases.     

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHO^P23H variant (hereafter called P23H) exhibited elevated levels of autophagy flux, and that pharmacological stimulation of autophagy accelerated retinal degeneration. In contrast, reducing autophagy flux pharmacologically or by rod-specific deletion of the autophagy-activating gene Atg5, improved photoreceptor structure and function. Furthermore, proteasome levels and activity were reduced in the P23H retina, and increased when Atg5 was deleted. Our findings suggest that autophagy contributes to photoreceptor cell death in P23H mice, and that decreasing autophagy shifts the degradation of misfolded RHO protein to the proteasome and is protective. These observations suggest that modulating the flux of misfolded proteins from autophagy to the proteasome may represent an important therapeutic strategy for reducing proteotoxicity in adRP and other diseases caused by protein folding defects.     

Increased Retinal Synthesis of Heparan Sulfate Proteoglycan and HNK-1 Glycoproteins Following Photoreceptor Degeneration

Abstract: In an earlier analysis of the retinal biosynthesis of proteoglycan, we noted that, following photoreceptor degeneration in the rd (retinal degeneration) mouse, the remaining inner retina exhibited a marked elevation in synthesis of heparan sulfate proteoglycan (HSPG), well above the level observed in the normal (nondegenerate) retina, as well as a pronounced increase in sulfation of protein substrates. Biochemical and autoradiographic results of ³⁵S-amino acid utilization reported here confirm that the ³⁵SO₄^2? differences seen previously are accompanied by increased protein synthesis in the rd retina. An intact photoreceptor cell layer is neither a barrier to nor a sink for the amino acid precursor. Further, we have examined sulfate utilization in four other rodent strains with photoreceptor degenerations. In each of the models examined, an increase in retinal synthesis of ³⁵SO₄^2?-labeled HSPG and glycoproteins occurs following photoreceptor degeneration. We have metabolically labeled with Na₂³⁵SO₄ isolated retinal cultures from the following: (a) mice with light-induced photoreceptor degeneration; (b) rd mice; (c) transgenic mice with photoreceptor degeneration; (d) RCS rats; and (e) rats with light-induced photoreceptor degeneration. Comparisons were made with concurrent cultures of control nondegenerate retinal tissues. Protein and proteoglycan-enriched fractions were prepared from the incubation media and guanidine HCI/detergent extracts of the retinas by ion-exchange chromatography. The ³⁵SO₄^2?-proteoglycans were identified by chondroitinase ABC and nitrous acid treatments. Retinas lacking photoreceptors produced at least five times the amount of ³⁵SO₄^2?-HSPG found in control incubations. The RCS and light-damaged rats also showed increased synthesis of ³⁵SO₄^2?-chondroitin sulfate proteoglycan relative to the control, though the increase was of lesser magnitude than the HSPG effect. ³⁵SO₄^2?-protein in degenerate and light-damaged retinas always contained at least twice the radioactivity found in comparable control preparations. The bulk of the increased radiolabeling was found in N-linked oligosaccharides, including several recognized by the HNK-1 antibody. These data suggest that a sustained increase in HSPG and HNK-1 glycoprotein synthesis is a consistent response of inner retinal cells following loss of photoreceptors and is independent of the cause of photoreceptor degeneration.     

Effective production of retinal from β-carotene using recombinant mouse β-carotene 15,15′-monooxygenase

The gene encoding β-carotene 15,15′-monooxygenase from Mus musculus (house mouse), which cleaves β-carotene into two molecules of retinal, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by His-tag affinity and resource Q ion exchange chromatography columns to a final specific activity of 0.51 U mg⁻¹. The optimum pH, temperature, substrate and detergent concentrations, and enzyme amount for effective retinal production were determined to be 9.0, 37°C, 200 mg l⁻¹ β-carotene, 5% (w/v) Tween 40, and 0.2 U ml⁻¹ enzyme, respectively. Under optimum conditions, the recombinant enzyme produced 72 mg l⁻¹ retinal in a 15-h reaction time, with a conversion yield of 36% (w/w). The specific activity of the purified enzyme and retinal production obtained in the present study were the highest results ever reported.     

Phenotypic analysis of boldit Galr1 knockout mice reveals a role for GALR1 galanin receptor in modulating seizure activity but not nerve regeneration

Summary The GALR1 galanin receptor is expressed at high levels within the central nervous system and is hypothesised to play a significant role in many of the central actions of galanin. To determine which specific actions of galanin are mediated by GALR1, we have developed mice that carry an insertional inactivating mutation within the first coding exon of the gene encoding GALR1 (Galr1). HomozygousGalr1 ^−/− mice are viable. Both male and female mice exhibit reduced circulating levels of insulin-like growth factor-I (IGF-I) but no significant difference in growth rate relative toGalr1 ^+/+ controls. Female homozygousGalr1 ^−/− mice are capable of breeding and nursing offspring. Functional recovery after sciatic nerve crush is not significantly different inGalr1 ^−/− mice relative toGalr1 ^+/+ controls, indicating that GALR1 does not mediate the nerve regenerative effects of galanin. However, homozygousGalr1 ^−/− mice exhibit spontaneous seizures, identifying a critical role for GALR1 in mediating the anti-seizure activity of galanin.     

Characterization of glycinergic synapses in vertebrate retinas

Summary Glycine is one of the essential neurotransmitters modulating visual signals in retina. Glycine activates Cl^- permeable receptors that conduct either inhibitory or excitatory actions, depending on the Cl⁻ electrical–chemical gradient (E _Cl) positive or negative to the resting potential in the cells. Interestingly, both glycine-induced inhibitory and excitatory responses are present in adult retinas, and the effects are confined in the inner and outer retinal neurons. Glycine inhibits glutamate synapses in the inner plexiform layer (IPL), resulting in shaping light responses in ganglion cells. In contrast, glycine excites horizontal cells and On-bipolar dendrites in the outer plexiform layer (OPL). The function of glycinergic synapse in the outer retina represents the effect of network feedback from a group of centrifugal neurons, glycinergic interplexiform cells. Moreover, immunocytochemical studies identify glycine receptor subunits (α1, α2, α3 and β) in retinas, forming picrotoxin-sensitive α-homomeric and picrotoxin-insensitive α/β-heteromeric receptors. Glycine receptors are modulated by intracellular Ca²⁺ and protein kinas C and A pathways. Extracellular Zn²⁺ regulates glycine receptors in a concentration-dependent manner, nanomolar Zn²⁺ enhancing glycine responses, and micromolar Zn²⁺ suppressing glycine responses in retinal neurons. These studies describe the function and mechanism of glycinergic synapses in retinas.     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the PRV recombinant virus TK⁻/gE⁻/GP5⁺ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine strain TK⁻/gE⁻/LacZ⁺, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK⁻/gE⁻/LacZ⁺, resulting in a PRV recombinant named TK⁻/gE⁻/GP5m⁺, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western blotting. TK⁻/gE⁻/GP5m⁺ and TK⁻/gE⁻/GP5⁺ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by TK⁻/gE⁻/GP5m⁺ against PRRSV were higher than that induced by TK⁻/gE⁻/GP5⁺. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV. Translated from Journal of Biotechnology, 2005, 21(6): 858–864 [译自: 生物工程学报]     

Changes in cell wall composition of deformedras1 − cells ofSchizosaccharomyces pombe

Disruption of theSchizosaccharomyces pombe ras1 gene results in a morphological transformation to large spheres, in contrast to wild-type cells which grow as rods. Chemical analysis of isolated cell walls showed no significant changes in saccharide content but an increase in protein and phosphate contents inras1 ⁻ walls relative to parent walls. Polymers tightly bound to the cell wall were solubilized by SDS treatment. Several compounds with molar mass ranging from 22 to 130 kDa and more were resolved by gel filtration and SDS-PAGE. Among low-molar-mass species, a component moving as a band at 31 kDa was conspicuous inras1 ⁻ cell walls. It was solubilized by heating in Tris-HCl buffer and shown to have a β-1,3-glucanase activity against laminarin. The level of the enzyme was by 30% higher in theras1 ⁻ cell wall than in the wild-type cell wall. This enzyme may participate in the remodelling of the rigid glucan network and account (at least partially) for the aberrant cell shape. Theras1 ⁻ cell wall contained a high level of charged polymers, especially phosphoproteins, raising the appealing possibility thatras1 ⁻ is involved in a putative kinase cascade required to sense and respond to external stimuli destined for the cell wall. Although the present study shows thatras1 loss of function and altered cell wall composition are closely linked defects, it has still to be shown that theras1 protein is directly involved in alterations found in the mutant cell walls.     

Enhanced anti-tumor activity of interferon-alpha in SOCS1-deficient mice is mediated by CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Interferon-alpha (IFN-α) is an immunomodulatory cytokine that is used clinically for the treatment of melanoma in the adjuvant setting. The cellular actions of IFN-α are regulated by the suppressors of cytokine signaling (SOCS) family of proteins. We hypothesized that the anti-tumor activity of exogenous IFN-α would be enhanced in SOCS1-deficient mice. SOCS1-deficient (SOCS1^−/−) or control (SOCS1^+/+) mice on an IFN-γ^−/− C57BL/6 background bearing intraperitoneal (i.p.) JB/MS murine melanoma cells were treated for 30 days with i.p. injections of IFN-A/D or PBS (vehicle). Log-rank Kaplan-Meier survival curves were used to evaluate survival. Tumor-bearing control SOCS1^+/+ mice receiving IFN-A/D had significantly enhanced survival versus PBS–treated mice (P = 0.0048). The anti-tumor effects of IFN-A/D therapy were significantly enhanced in tumor-bearing SOCS1^−/− mice; 75% of these mice survived tumor challenge, whereas PBS-treated SOCS1^−/− mice all died at 13-16 days (P = 0.00038). Antibody (Ab) depletion of CD8⁺ T cells abrogated the anti-tumor effects of IFN-A/D in SOCS1^−/− mice as compared with mice receiving a control antibody (P = 0.0021). CD4⁺ T-cell depletion from SOCS1^−/− mice also inhibited the effects of IFN-A/D (P = 0.0003). IFN-A/D did not alter expression of CD80 or CD86 on splenocytes of SOCS1^+/+ or SOCS1^−/− mice, or the proportion of T regulatory cells or myeloid-derived suppressor cells in SOCS1^+/+ or SOCS1^−/− mice. An analysis of T-cell function did reveal increased proliferation of SOCS1-deficient splenocytes at baseline and in response to mitogenic stimuli. These data suggest that modulation of SOCS1 function in T-cell subsets could enhance the anti-tumor effects of IFN-α in the setting of melanoma.     

Absence of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM) interferes with the maintenance,but not with the formation of peripheral myelin

We have previously shown that mice deficient in the gene for the myelin-associated glycoprotein (MAG) develop normal myelin in the peripheral nerves, but show axon and myelin degeneration at eight months of age, suggesting that MAG is involved in the maintenance of axon-Schwann cell integrity. The search for molecules that might replace MAG during myelination revealed an overexpression of the neural cell adhesion molecule (N-CAM) at those aspects where MAG is detectable in wild type mice. To test whether N-CAM might compensate for MAG during myelination in MAG-deficient mice, double mutants deficient in both MAG and N-CAM (MAG⁻/N-CAM⁻mice) were generated by cross-breeding the single mutants. Whereas alterations of myelin development were not detectable in either of the single or double mutants, degeneration of myelin and axons occurred approximately 4 weeks earlier in MAG⁻/N-CAM⁻than in MAG⁻mutants. Furthermore, at 8 weeks of age, single fiber preparation and electron microscopy revealed that the number of profiles indicative of degeneration was substantially increased in MAG⁻/N-CAM⁻mutants when compared to MAG⁻mice. These data suggest that in MAG-deficient mice N-CAM does not compensate for MAG in myelin formation but partially substitutes for it in the maintenance of axon-myelin integrity. Received: 20 May 1996 / Accepted: 19 July 1996     

Tribbles homolog 3-mediated targeting the AKT/mTOR axis in mice with retinal degeneration

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3^−/− retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.Subject terms: Translation, Diseases     

Q344ter Mutation Causes Mislocalization of Rhodopsin Molecules That Are Catalytically Active: A Mouse Model of Q344ter-Induced Retinal Degeneration

Q344ter is a naturally occurring rhodopsin mutation in humans that causes autosomal dominant retinal degeneration through mechanisms that are not fully understood, but are thought to involve an early termination that removed the trafficking signal, QVAPA, leading to its mislocalization in the rod photoreceptor cell. To better understand the disease mechanism(s), transgenic mice that express Q344ter were generated and crossed with rhodopsin knockout mice. Dark-reared Q344ter^rho+/− mice exhibited retinal degeneration, demonstrating that rhodopsin mislocalization caused photoreceptor cell death. This degeneration is exacerbated by light-exposure and is correlated with the activation of transducin as well as other G-protein signaling pathways. We observed numerous sub-micrometer sized vesicles in the inter-photoreceptor space of Q344ter^rho+/− and Q344ter^rho−/− retinas, similar to that seen in another rhodopsin mutant, P347S. Whereas light microscopy failed to reveal outer segment structures in Q344ter^rho−/− rods, shortened and disorganized rod outer segment structures were visible using electron microscopy. Thus, some Q344ter molecules trafficked to the outer segment and formed disc structures, albeit inefficiently, in the absence of full length wildtype rhodopsin. These findings helped to establish the in vivo role of the QVAPA domain as well as the pathways leading to Q344ter-induced retinal degeneration.     

Studies on inositol-mediated expression of MAL gene encoding maltase and phospholipid biosynthesis in Schizosaccharomyces pombe

In this study, the effects of inositol addition on expression of the MAL gene encoding maltase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe (a naturally inositol-requiring strain) were examined. We found that specific maltase activity was at its maximum when the concentration of added inositol reached 6 μg ml⁻¹ in a synthetic medium containing 2.0% (w/v) glucose. When the concentration of added inositol was 1 μg ml⁻¹ in the medium, repression of MAL gene expression occurred at glucose concentration higher than 0.2% (w/v). However, when S. pombe was cultured in the synthetic medium containing 6 μg ml⁻¹, repression of maltase gene expression occurred only at initial glucose concentration above 1.0% (w/v). More mRNA encoding maltase was detected in the cells grown in the medium with 6 μg ml⁻¹ inositol than in those grown in the same medium with 1 μg ml⁻¹ inositol. These results demonstrate that higher inositol concentrations in the synthetic medium could derepress MAL gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 6 μg ml⁻¹ of inositol was higher than that of the yeast cells grown in the same medium with 1 μg ml⁻¹ of inositol. This means that PI may be involved in the derepression of MAL gene expression in S. pombe.