Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Vacuolar-type H<Superscript>+</Superscript>-ATPase with the <Emphasis Type="Italic">a</Emphasis>3 isoform is the proton pump on premature melanosomes

The melanosome, an organelle specialized for melanin synthesis, is one of the lysosome-related organelles. Its lumen is reported to be acidified by vacuolar-type H⁺-ATPase (V-ATPase). Mammalian V-ATPase exhibits structural diversity in its subunit isoforms; with regard to membrane intrinsic subunit a, four isoforms (a1–a4) have been found to be localized to distinct subcellular compartments. In this study, we have shown that the a3 isoform is co-localized with a melanosome marker protein, Pmel17, in mouse melanocytes. Acidotropic probes (LysoSensor and DAMP) accumulate in non-pigmented Pmel17-positive melanosomes, and DAMP accumulation is sensitive to bafilomycin A1, a specific inhibitor of V-ATPase. However, none of the subunit a isoforms is associated with highly pigmented mature melanosomes, in which the acidotropic probes are also not accumulated. oc/oc mice, which have a null mutation at the a3 locus, show no obvious defects in melanogenesis. In the mutant melanocytes, the expression of the a2 isoform is modestly elevated, and a considerable fraction of this isoform is localized to premature melanosomes. These observations suggest that the V-ATPase keeps the lumen of premature melanosomes acidic, whereas melanosomal acidification is less significant in mature melanosomes. Ge-Hong Sun-Wada and Yoh Wada contributed equally to this study. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Hayashi and Noda Foundations.     

The Increase in Retinal Cells Proliferation Induced by FGF2 is Mediated by Tyrosine and PI3 Kinases

Since 1973, multiple effects of basic fibroblast growth factor have been described in a large number of cells. These effects include proliferation, survival and differentiation. The aim of this work was to study the intracellular pathways involved in the basic fibroblast growth factor (FGF2) effect on rat retinal cells proliferation in vitro. Our data show that treatment with FGF2 increases proliferation in a concentration- and time-dependent manner. The effect of 25 ng/ml FGF2 was blocked by 10 μM genistein, a tyrosine kinase inhibitor and by 25 μM LY294002, a PI3 kinase inhibitor. The concomitant treatment with 0.3 μM chelerythrine chloride, a protein kinase C inhibitor, and 6.25 μM LY294002 also inhibited the effect of FGF2. Our results suggest that the proliferative effect of FGF2 on retinal cell cultures involves the activation of distinct kinases.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arginine Uptake in Normal and Diabetic Rat Retina and Retinal Pigment Epithelium

Diabetes-induced increase in oxidative stress is postulated as playing a significant role in the development of retinopathy. The retinal pigment epithelium (RPE) which forms part of the retinal blood barrier has been reported to be affected in diabetes. Besides functioning as a neurotransmitter, the radical nitric oxide (NO) can act as a cytotoxic agent. NO is synthesized by nitric oxide synthase (NOS) that oxidizes arginine to citrulline producing NO. Given that intracellular concentration of arginine depends mainly on its transport, we studied arginine transport in RPE and retina from normal and streptozotocin-induced diabetic rats. Retina and RPE take up arginine by a saturable system with an apparent K_M of 70–80 μM. Tissue incubation in the presence of insulin or high glucose concentrations significantly increased arginine transport in RPE but not in retina from control rats. Similarly, arginine uptake was enhanced in RPE, but not in the retina from streptozotocin-induced diabetic rats. However, NO content was two-fold higher in diabetic retina and RPE compared to that in the control rats. Such findings may suggest that diabetes induced an increase in NO levels in RPE, which may have brought about alterations in its functioning and in turn manifestations of diabetic retinopathy. Special issue article in honor of Dr. Ricardo Tapia.     

Neurite responses to ephrin-A5 modulated by BDNF: evidence for TrkB-EphA interactions

In the developing visual system, growing retinal ganglion cell (RGC) axons are exposed to multiple guidance and growth factors. Furthermore, the relative levels of these factors are differentially regulated as topography is roughly established and then refined. We have shown that during the establishment of rough topography (P3), growth cones of pure and explanted RGCs treated with combinations of BDNF and ephrin-A5-Fc responded differently than RGCs treated with BDNF or ephrin-A5-Fc alone (p = 0.0083). The response to the combined treatment mimicked that of RGCs cultured with ephrin-A5-Fc alone once topography refines. The guidance cue receptors EphA and TrkB were shown to co-localise in RGCs in vitro. Furthermore, EphA and TrkB receptors interacted directly in in vitro binding assays. Our results suggest that the conversion of growth cone responses from collapse to stabilisation as topography refines, occurs as a result of interactions between EphA and TrkB receptors.     

The vagus nerve, food intake and obesity

Food interacts with sensors all along the alimentary canal to provide the brain with information regarding its composition, energy content, and beneficial effect. Vagal afferents innervating the gastrointestinal tract, pancreas, and liver provide a rapid and discrete account of digestible food in the alimentary canal, as well as circulating and stored fuels, while vagal efferents, together with the sympathetic nervous system and hormonal mechanisms, codetermine the rate of nutrient absorption, partitioning, storage, and mobilization. Although vagal sensory mechanisms play a crucial role in the neural mechanism of satiation, there is little evidence suggesting a significant role in long-term energy homeostasis. However, increasing recognition of vagal involvement in the putative mechanisms making bariatric surgeries the most effective treatment for obesity should greatly stimulate future research to uncover the many details regarding the specific transduction mechanisms in the periphery and the inter- and intra-neuronal signaling cascades disseminating vagal information across the neuraxis.     

Enhanced Survival of Melanopsin-expressing Retinal Ganglion Cells After Injury is Associated with the PI3 K/Akt Pathway

In the present study, we studied the factors that contribute to the injury-resistant property of melanopsin-expressing retinal ganglion cells (mRGCs). Since phosphatidylinositol-3 kinase (PI3 K)/Akt signaling pathway is one of the well-known pathways for neuronal cell survival, we investigated the survival of mRGCs by applying the PI3 K/Akt specific inhibitors after injury. Two injury models, unilateral optic nerve transection and ocular hypertension, were adopted using Sprague-Dawley rats. Inhibitors of PI3 K/Akt were injected intravitreally following injuries to inhibit the PI3 K/Akt signaling pathway. Retinas were dissected after designated survival time, immunohistochemistry was carried out to visualize the mRGCs using melanopsin antibody and the number of mRGCs was counted. Co-expression of melanopsin and phospho-Akt (pAkt) was also examined. Compared to the survival of non-melanopsin-expressing RGCs, mRGCs showed a marked resistance to injury and co-expressed pAkt. Application of PI3 K/Akt inhibitors decreased the survival of mRGCs after injury. Our previous study has shown that mRGC are less susceptible to injury following the induction of ocular hypertension. In this study, we report that mRGCs were injury-resistant to a more severe type of injury, the optic nerve transection. More importantly, the PI3 K/Akt pathway was found to play a role in maintaining the survival of mRGCs after injury.     

正常猕猴与人视网膜血管的比较

目的比较正常猕猴与人视网膜血管的异同,为进一步利用猕猴建立动物模型来研究视网膜血管打下基础。方法取健康成年猕猴眼球6只和人角膜移植供体剩余眼杯8只的视网膜,用ADP酶法进行血管染色,对两者视网膜血管的走行、血管分级、毛细血管分层以及黄斑区血管拱环等进行比较,测量结果进行统计学检验。结果猕猴与人的视网膜铺片经ADP酶法染色后见视网膜血管自穿出视盘后的一级血管逐渐分支变细,直至五级血管即毛细血管;在视盘旁、赤道部、周边部两者血管面积百分比没有差异;视盘旁血管分为多层,赤道部有两层,且深浅层间相互交通,周边部仅见一层毛细血管且较稀疏;两者黄斑区毛细血管均较密集,有形态完整呈不规则状的血管拱环,血管面积百分比以及血管拱环的面积、周长和直径没有差异。结论猕猴与人在视网膜血管走行、分级、毛细血管分层以及黄斑区血管拱环等多方面有良好的相似性,可用作人类视网膜血管、尤其是黄斑区视网膜血管研究的良好动物模型。     

血塞通对糖尿病视网膜病变患者视网膜微循环的影响

目的:探究血塞通对糖尿病视网膜病变(DR)患者视网膜微循环的影响。方法:收集2013年2月至2016年2月在我院接受治疗的95例双眼糖尿病视网膜病变患者,并随机分为对照组(48例)和观察组(47例)。对照组患者给予常规降糖治疗,观察组在对照组的基础上给予血塞通治疗。观察比较两组治疗前后的视网膜微循环指标,包括视网膜中央动脉(CRA)的舒张末期血流速度(EDV)、收缩期峰值流速(PSV)及阻力指数(RI)以及视网膜中央静脉(CRV)的最低血流速度(Vmin)、最高血流速度(Vmax)及平均血流速度(MV),血液流变学指标包括全血低切黏度(NBL)、全血高切黏度(NBH)、红细胞变形指数(DE)、红细胞压积(Hct)、红细胞聚集指数(AE)及血沉(ESR)以及临床疗效。结果:治疗后,观察组的临床总有效率为87.8%,明显高于对照组的61.4%(P0.05),对照组的EDV、Vmax、Vmin均较治疗前明显改善(P0.05),NBL和NBH较治疗前明显降低,且观察组的PSV、EDV明显高于对照组,而MV、RI、Vmax、Vmin均低于对照组(P0.05),NBL、NBH、DE、Hct、AE及ESR均较治疗前明显改善,且相较于对照组改善幅度更显著(P0.05)。结论:血塞通联合常规降糖治疗糖尿病视网膜病变患者的疗效显著,可有效改善患者的视网膜微循环和血液流变学。     

Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization

Phototaxis in the unicellular green alga Chlamydomonas reinhardtii is mediated by rhodopsin-type photoreceptor(s). Recent expressed sequence tag database from the Kazusa DNA Research Institute has provided the basis for unequivocal identification of two archaeal-type rhodopsins in it. Here we demonstrate that one is located near the eyespot, wherein the photoreceptor(s) has long been thought to be enriched, along with the results of bioinformatic analyses. Secondary structure prediction showed that the second putative transmembrane helices (helix B) of these rhodopsins are rich in glutamate residues, and homology modeling suggested that some additional intra- or intermolecular interactions are necessary for opsin-like folding of the N-terminal ca. 300-aa membrane spanning domains of 712 and 737-aa polypeptides. These results complement physiological and electrophysiological experiments combined with the manipulation of their expression [O.A. Sineshchekov, K.H. Jung, J.H. Spudich, Proc. Natl. Sci. USA 99 (2002) 8689; G. Nagel, D. Olig, M. Fuhrmann, S. Kateriya, A.M. Musti, E. Bamberg, P. Hegemann, Science 296 (2002) 2395].