First Report of Banana bract mosaic virus in 'Cavendish' Banana in Ecuador.

Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of bract mosaic disease. The disorder has been considered a serious constraint to banana and plantain production in India and the Philippines, where the virus was first identified (3). To date, the presence of BBrMV has been reported only in a few banana-growing countries in Asia (3). In the Americas, BBrMV has been detected by ELISA tests in Colombia only (1). The efficient spread of BBrMV through aphids and vegetative material increases the quarantine risk and requires strict measures to prevent entrance of the virus to new areas. In Ecuador-the world's number one banana exporter-the banana industry represents the main agricultural income source. Thus, early detection of banana pathogens is a priority. In June of 2012, mosaic symptoms in bracts and bunch distortion of 'Cavendish' banana were observed in a commercial field in the province of Guayas, Ecuador. Leaves from 35 symptomatic plants were tested for Cucumber mosaic virus (CMV), Banana streak virus (BSV), and BBrMV using double antibody sandwich ELISA kits from Adgen (Scotland, UK). Twenty-one plants tested positive for BBrMV but not for CMV or BSV. In order to confirm the ELISA results, fresh or lyophilized leaf extracts were used for immunocapture reverse transcription (IC-RT)-PCR. In addition, total RNA was extracted from the ELISA-positive samples and subjected to RT-PCR. The RT reactions were done using both random and oligo dT primers. Several sets of primers, flanking conserved regions of the virus coat protein (CP), have been used for PCR-detection of BBrMV (2,3,4). The Ecuadorian BBrMV isolate was successfully detected by three primer sets with reported amplification products of 324, 280, and 260 nucleotides long, respectively (3,4). Amplification products of the expected size were purified and sequenced. All the nucleotide sequences obtained from 20 PCR-positive symptomatic plants were 100% identical between each other. However, 99% identity was observed when PCR products from the Ecuadorian isolate were compared with the corresponding fragment of a BBrMV isolate from the Philippines (NCBI Accession No. DQ851496.1). PCR products of the Ecuadorian isolate, amplified by the different CP primers described above, were assembled into a 408-bp fragment and deposited in the NCBI GenBank (KC247746). Further testing confirmed the presence of BBrMV in symptomatic plants from four different provinces. To our knowledge, this is the first report of BBrMV in Ecuador and the first BBrMV partial nucleotide sequence reported from the Americas. It is worth mentioning that primer set Bract 1/Bract 2, which amplifies a 604-bp product (2), was not effective in detecting the Ecuadorian isolate. It is hypothesized that nucleotide variation at the reverse primer site is the cause of the lack of amplification with this primer set, since the forward primer is part of the sequenced product and no variation was found. Sequencing of the entire CP region is underway to conduct phylogenetic analysis and determine genetic relationships across several other BBrMV isolates. References: (1) J. J. Alarcon et al. Agron 14:65, 2006. (2) M. F. Bateson and J. L. Dale. Arch. Virol 140:515, 1995. (3) E. M. Dassanayake. Ann. Sri Lanka Dept. Agric. 3:19, 2001. (4) M. L. Iskra-Caruana et al. J. Virol. Methods 153:223, 2008.

First Report of Raspberry bushy dwarf virus in Andean Blackberry (Rubus glaucus) in Central Ecuador.

During the past two decades, several viruses have been identified from Rubus spp. in wild and commercial plantings around the world (2). In Ecuador, approximately 14 tons of blackberries are produced each year from an estimated area of 5,500 ha. In 2012, a preliminary survey was conducted to determine the presence of RNA viruses in Rubus glaucus, the most prevalent blackberry in Ecuador. Fifteen plants showing leaf mottling and severe mosaic were leaf-sampled from each of five different fields in Azuay Province. A total of 12 pooled samples of 20 g were obtained from the collected symptomatic tissue and used for dsRNA extraction using a cellulose-based protocol for detection of RNA viruses in plants (3). Three dsRNA segments of approximately 5 kbp, 2 kbp, and 900 bp were observed from all 12 dsRNA preparations. The dsRNA was heat-denatured and used as template for the generation of cDNA library using the universal random primer 5'-GCCGGAGCTCTGCAGAATTCNNNNNN-3', for reverse transcription (RT), and the anchor primer 5'-GCCGGAGCTCTGCAGAATTC-3'for PCR as described (1). The PCR products were cloned using a StrataClone Kit (Agilent, CA) and sequenced (Macrogen, Korea). Sequence analysis revealed the presence of Raspberry bushy dwarf virus (RBDV), a pollen-borne Idaeovirus naturally found in several Rubus spp. worldwide. Approximately 120 RBDV sequences obtained from the Ecuadorean isolate were assembled into two contigs belonging to RNA1 and RNA2. Both sequences were re-confirmed by RT-PCR using specific primers. Partial sequences were assigned GenBank Accessions KC315894, KC315893, and KC315892 for the replicase, MP and CP, respectively. Furthermore, BLAST searches showed that the nucleotide sequence corresponding to the replicase was 95% similar to an isolate from the resistance breaking R15 strain (S51557.1), whereas the MP and CP nucleotide sequences were up to 98% similar to a Slovenian isolate (EU796088.1). Primers designed to amplify a 427-bp portion of the CP were used to detect RBDV from four blackberry plantings in two distant production areas: Ambato in Tungurahua Province and Paute in Azuay Province. Leaf mottling and severe mosaic was observed in 90% of blackberry fields in those two locations. Leaf samples (n = 90) were randomly collected from both symptomatic and asymptomatic plants in each location. In Ambato, RBDV was detected in 50% and 40% of symptomatic and asymptomatic plants, respectively. In Paute, RBDV was present in 70% of symptomatic plants and 29% of asymptomatic plants. The presence of RBDV in asymptomatic plants suggests the virus might not be the sole causal agent of the disorder. Further studies are needed to determine the role of RBDV in the observed symptoms, since virus complexes responsible for increased severity of symptoms have been commonly reported in Rubus spp. (4). R. glaucus is native to the tropical highlands (from Ecuador to Mexico) and differs from blackberries commercially grown in the United States and Europe. Therefore, RBDV-induced symptoms reported in blackberry grown in the United States and Europe may not be extrapolated to the Andes berry. To the best of our knowledge, this is the first report of RBDV from blackberry in Ecuador. References: (1) P. Froussard. Nucleic Acids Res. 20:2900, 1992. (2) R. R. Martin et al. Plant Dis. 97:168, 2013. (3). T. J. Morris and J. A. Dodds. Phytopathology 69:854. 1979. (4) D. F. Quito-Avila et al. J. Virol. Methods 179:38, 2012.

Umbilical Cord-derived Mesenchymal Stem Cells for COVID-19 Patients with Acute Respiratory Distress Syndrome (ARDS).

The coronavirus SARS-CoV-2 is cause of a global pandemic of a pneumonia-like disease termed Coronavirus Disease 2019 (COVID-19). COVID-19 presents a high mortality rate, estimated at 3.4%. More than 1 out of 4 hospitalized COVID-19 patients require admission to an Intensive Care Unit (ICU) for respiratory support, and a large proportion of these ICU-COVID-19 patients, between 17% and 46%, have died. In these patients COVID-19 infection causes an inflammatory response in the lungs that can progress to inflammation with cytokine storm, Acute Lung Injury (ALI), Acute Respiratory Distress Syndrome (ARDS), thromboembolic events, disseminated intravascular coagulation, organ failure, and death. Mesenchymal Stem Cells (MSCs) are potent immunomodulatory cells that recognize sites of injury, limit effector T cell reactions, and positively modulate regulatory cell populations. MSCs also stimulate local tissue regeneration via paracrine effects inducing angiogenic, anti-fibrotic and remodeling responses. MSCs can be derived in large number from the Umbilical Cord (UC). UC-MSCs, utilized in the allogeneic setting, have demonstrated safety and efficacy in clinical trials for a number of disease conditions including inflammatory and immune-based diseases. UC-MSCs have been shown to inhibit inflammation and fibrosis in the lungs and have been utilized to treat patients with severe COVID-19 in pilot, uncontrolled clinical trials, that reported promising results. UC-MSCs processed at our facility have been authorized by the FDA for clinical trials in patients with an Alzheimer's Disease, and in patients with Type 1 Diabetes (T1D). We hypothesize that UC-MSC will also exert beneficial therapeutic effects in COVID-19 patients with cytokine storm and ARDS. We propose an early phase controlled, randomized clinical trial in COVID-19 patients with ALI/ARDS. Subjects in the treatment group will be treated with two doses of UC-MSC (l00 × 106 cells). The first dose will be infused within 24 hours following study enrollment. A second dose will be administered 72 ± 6 hours after the first infusion. Subject in the control group will receive infusion of vehicle (DPBS supplemented with 1% HSA and 70 U/kg unfractionated Heparin, delivered IV) following the same timeline. Subjects will be evaluated daily during the first 6 days, then at 14, 28, 60, and 90 days following enrollment (see Schedule of Assessment for time window details). Safety will be determined by adverse events (AEs) and serious adverse events (SAEs) during the follow-up period. Efficacy will be defined by clinical outcomes, as well as a variety of pulmonary, biochemical and immunological tests. Success of the current study will provide a framework for larger controlled, randomized clinical trials and a means of accelerating a possible solution for this urgent but unmet medical need. The proposed early phase clinical trial will be performed at the University of Miami (UM), in the facilities of the Diabetes Research Institute (DRI), UHealth Intensive Care Unit (ICU) and the Clinical Translational Research Site (CTRS) at the University of Miami Miller School of Medicine and at the Jackson Memorial Hospital (JMH).

The visual cycle operates via an isomerase acting on all-trans retinol in the pigment epithelium.

Thirty years have elapsed since Wald and his colleagues showed that 11-cis retinal was isomerized to all-trans when rhodopsin was bleached, yet little has been understood about the reverse process that generates 11-cis retinal for rhodopsin regeneration. It is not known whether the isomerization is enzyme-mediated, whether it occurs in the pigment epithelium or in the retina, or whether retinal, retinol, or a retinyl ester is the vitamin A compound that is isomerized. Radiolabeled all-trans retinol and high-performance liquid chromatography have now been used to demonstrate the existence of an eye-specific, membrane-bound enzyme (retinol isomerase) that converts all-trans to 11-cis retinol in the dark. Retinol isomerase is concentrated in the pigment epithelium; this localization clarifies the role of this tissue in rhodopsin regeneration and explains the need to transfer all-trans retinol from the rod outer segments to the pigment epithelium during the visual cycle.

Quantum Yield for Production of CH3NC in the Photolysis of CH3NCS.

The ultraviolet spectrum of methyl isothiocyanate (CH(3)NCS) and the quantum yield for its dissociation into methyl isocyanide (CH(3)NC) and atomic sulfur at 308 nanometers, Phi = 0.98 +/- 0.24, were measured. Methyl isothiocyanate is widely used as an agricultural fumigant and readily enters the atmosphere during and after application. The results indicate that photodissociation by sunlight is an effective pathway for its removal from the atmosphere.

N-terminal sequence homologies in interstitial retinol-binding proteins from 10 vertebrate species.

We report here the first comprehensive comparative NH2-terminal sequence studies of interstitial retinol-binding protein (IRBPs) from nine mammals (including cattle) and one amphibian. This study has revealed that in many species the N-terminus of IRBP includes a 3-6 amino acid extension. IRBP possessing this leader sequence is sometimes mixed with IRBP from which this sequence has been excised.

Levels of alpha- and gamma-tocopherol in human eyes: evaluation of the possible role of IRBP in intraocular alpha-tocopherol transport.

Alpha-tocopherol was distributed almost equally between the retina and its underlying pigmented layers (pigment epithelium and choroid). Only 8.4% of the total alpha-tocopherol occurred in the iris and ciliary body. Alpha-tocopherol content was expressed as amount per eye, per cm2, and per 100 g wet weight. The combined retina and pigment epithelium-choroid contained 2.9 +/- 1.0 mg/100 g wet weight (means +/- SD, n = 30 donors). Gamma-tocopherol represented 20.9 +/- 12.2 mol % of the alpha-tocopherol. The anterior tissues contained 0.4 +/- 0.2 mg/100 g (n = 19 donors). No significant correlation with age was found. Purified bovine interstitial retinol-binding protein (IRBP) bound exogenous 3H-alpha-tocopherol, which could be displaced by unlabeled all-trans retinol (KD = 10(-6) M). Much higher concentrations of unlabeled alpha-tocopherol were required to achieve a partial displacement of bound 3H-all-trans retinol. No endogenous alpha-tocopherol could be detected in bovine interphotoreceptor matrix.

Vitamin A in human eyes: amount, distribution, and composition.

The amount, distribution, and composition of vitamin A stored in the eyes of 29 postmortem donors was determined by a combination of techniques, including high-pressure liquid chromatography. The vitamin A concentration in the pigment epithelium-choroid (RPE-Ch) was the highest observed for human non-liver tissue and amounted to 7.9 +/- 4.3 nmol/eye (n = 28), or 10.4 +/- 7.1 microgram/gm (n = 27). There was no evidence for significant losses during the interval between death and enucleation or during subsequent storage at 4 degrees C. The vitamin A extracted from the retina was 15.3% of that in the corresponding RPE-Ch. By measuring rhodopsin regeneration in retinal homogenates incubated with 11-cis retinal, we estimated that the amount of vitamin A in the RPE-Ch of fully dark-adapted eyes would represent 2.5 mole equivalents of the retinal rhodopsin, a value similar to that found in the frog. A preponderance of the vitamin A in the eye was esterified (98.3% in the RPE-Ch, 79.3% in the retina) and consisted principally of stearate and palmitate in the ratio of 1:4.8. A small amount of oleate was also detected. The ratio of 11-cis isomer over the all-trans averaged 1.52 +/- 0.48 (n - 11). Variable, usually small proportions of 13-cis retinyl esters were also present. Intact RPE-Ch or isolated RPE cells esterified exogenous all-trans-3H2-retinol to the same fatty acids in roughly the same proportions as in the endogenous stores. The all-trans configuration was mainly retained during uptake and esterification, although some isomerization to 13-cis also occurred. No 11-cis isomer was formed under these conditions.

High-pressure liquid chromatography of fatty acid esters of retinol isomers. Analysis of retinyl esters stored in the eye.

We have synthesized the all-trans, 13-cis, 11-cis, and 9-cis retinyl esters of some or all of the following fatty acids: caprylic (8:0), capric (10:0), lauric (12:0), myristic (14:0), palmitic (16:0), stearic (18:0), arachidic (20:0), palmitoleic (16:1), oleic (18:1), linoleic (18:2), linolenic (18:3), arachidonic (20:4), and docosahexaenoic (22:6). Mixtures of these compounds were analyzed by high-pressure liquid chromatography on an Altex 100 chromatograph. This approach has enabled us to investigate and identify both the isomeric form and fatty acid basis of the retinyl esters present in rabbit and rat ocular tissues. Dark-adapted rabbits had 11-cis and all-trans retinyl esters, whereas light-adapted albino rats had only the all-trans isomer. The fatty acids utilized for esterification were almost wholly palmitic for the rabbit, whereas both stearic and palmitic were present in the rat esters. Rabbits are similar to frogs in that a large proportion (80%) of their ocular retinyl esters occurred in large, fluorescent oil droplets in the pigment epithelium. These are absent in rats.

Docosapentaenoic acid is converted to docosahexaenoic acid in the retinas of normal and prcd-affected miniature poodle dogs.

PURPOSE: Docosahexaenoic acid (DHA, 22:6n-3) is the major fatty acid of photoreceptor membranes that is necessary for optimal retinal function. Miniature poodle dogs with progressive rod-cone degeneration have lower plasma levels of DHA than normal dogs and higher 22:5n-3/22:6n-3 ratios. The purpose of this study was to test the hypothesis that the metabolic defect in dogs affected with progressive rod-cone degeneration was a reduced capacity for ocular synthesis of DHA from its precursor 22:5n-3. METHODS: The in vivo retinal conversion of [14C]22:5n-3 to [14C]22:6n-3 was investigated by injecting normal dogs and dogs affected with progressive rod-cone degeneration intravitreally with [14C]22:5n-3. After 72 hours, rod outer segments, remaining retina, and retinal pigment epithelium/choroid were analyzed for metabolic products. RESULTS: Using high-performance liquid chromatography, six radioactive peaks were detected in both normal and affected dogs: [14C]20:5n-3, [14C]22:6n-3, [14C]22:5n-3, [14C]24:6n-3, [14C]24:5n-3, and [14C]18:0. The majority of the label in each tissue was in 22:6n-3 and there was no difference in the dpm% of [14C]22:6n-3 in normal and affected animals in any of the three tissues. Voss et al (J Biol Chem 1991;266:19995-20000) proposed a new pathway for the synthesis of DHA that involves elongation of 22:5n-3 to 24:5n-3, desaturation to 24:6n-3, and beta-oxidation to 22:6n-3. Identification of the products [14C]24:5n-3 and [14C]24:6n-3 verified that these putative intermediates are present in the dog retina. CONCLUSIONS: The finding of large amounts of label in DHA shows that the normal and progressive rod-cone-degenerated retina and retinal pigment epithelium are capable of DHA synthesis from 22:5n-3. The presence of radioactivity in 24:5n-3 and 24:6n-3 suggests that the synthesis of DHA in the retina is similar to that described in the liver.

Rhodopsin, vitamin A, and interstitial retinol-binding protein in the rd chicken.

In order to determine whether blindness in the rd strain of Rhode Island Red chickens is due to a defect in the vitamin A (visual) cycle, spectroscopy, high performance liquid chromatography, and immunochemical techniques were used to compare the amounts of rhodopsin, interstitial retinol-binding protein, and vitamin A compounds in the dark-adapted eyes of homozygous rd and heterozygous carriers. In both groups of chickens, (up to 6 weeks post-hatching) the distribution of stored vitamin A differed from other vertebrates (mammals, amphibians, fish) in that more than half of the retinyl palmitate/stearate occurred in the neurosensory retina. The 11-cis isomer accounted for nearly 100% of the retinyl palmitate/stearate in the neurosensory retinas of both groups. In the pigmented layers (pigment epithelium and choroid) the 11-cis isomer amounted to 70.1 +/- 4.2% in the carrier, and 65.1 +/- 2.9% in the rd birds. With respect to their content of rhodopsin, IRBP, retinyl palmitate/stearate and unesterified retinol, (both 11-cis and all-trans isomers) no significant difference could be demonstrated between the eyes of rd and carrier chickens (3 days and 28 days post-hatching). These results therefore demonstrate that the ocular tissues of rd chickens do not lack IRBP, the putative extracellular transport protein for vitamin A, that these tissues synthesize and store the 11-cis isomer of vitamin A, and that the 11-cis isomer is used to form rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipids of human retina, retinal pigment epithelium, and Bruch's membrane/choroid: comparison of macular and peripheral regions.

PURPOSE: To understand the difference between macular and peripheral regions, tissue samples of human retina, retinal pigment epithelium, and Bruch's membrane/choroid were dissected and analyzed for lipid composition. METHOD: To facilitate dissections and enhance the recovery of tissues, eyecups were prefixed for 1 hour in 10% formalin (pH 7). Lipids were extracted from 4-mm trephined punches of tissues and analyzed by high-performance liquid chromatography. After separation of neutral lipids and phospholipids, total fatty acids in both lipid classes were quantitated. RESULTS: The major phospholipid classes in retina, retinal pigment epithelium, and Bruch's membrane/choroid were phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidyl serine; the major fatty acids were palmitic (16:0), stearic (18:0), and oleic (18:1). Although the three tissues had similar total fatty acid and phospholipid components, their relative compositions were different. Neutral lipid/phospholipid ratios in retinal pigment epithelium and Bruch's membrane/choroid were almost three times higher than in the retina. CONCLUSIONS: This study provides information about the lipid composition of macular and peripheral regions of the human retina, retinal pigment epithelium, and Bruch's membrane/choroid. The methodology employed enabled study of lipids in small amounts of tissue, which will be of value in investigating the biochemical aspects of age-related macular degeneration.

Vitamin A utilization in human retinal pigment epithelial cells in vitro.

Vitamin A (vit A) metabolism was studied in freshly isolated and cultured human retinal pigment epithelial (RPE) cells obtained from postmortem donor eyes. Fluorometric determination of vit A in human RPE cells demonstrated that freshly isolated cells contained approximately 1.0 to 4.0 pg vit A/cell which decreased with increasing time in culture; after 48 hrs in culture cellular vit A was reduced 80%. High performance liquid chromatography (hplc) profiles of the retinyl esters in freshly isolated RPE cells showed the presence of 11-cis retinyl stearate and palmitate and all-trans retinyl stearate, palmitate and oleate; all-trans palmitate was the major ester. Hplc analyses of cell cultures supplemented with all-trans retinol, using fatty acid-free bovine serum albumin as a carrier, showed that the cells in primary and subcultures took up all-trans retinol and esterified it to form palmitate, stearate, and oleate. Palmitate was the major ester synthesized by the cells in primary cultures. In the subcultures the esters synthesized differed from that found in freshly isolated cells and in the cells in primary culture; in the subcultures, the overall synthesis of ester was reduced and oleate was more prominent. The esters that were synthesized in culture were all-trans; the formation of 11-cis isomers was not observed in human RPE cells in culture. Electron microscopy of retinol-supplemented cultures indicated that vit A doses up to 1.0 micrograms/ml had no obvious effects on the cells; at higher doses the cells no longer adhered to the culture surface.

Vitamin A and interstitial retinol-binding protein in an eye with recessive retinitis pigmentosa.

The composition and amount of vitamin A stored in the retinal pigment epithelium and choroid (RPE-Ch) was evaluated in postmortem donor eyes from a patient with retinitis pigmentosa that was probably inherited by an autosomal recessive mode. Additionally, the soluble proteins in the neural retina and RPE-Ch cytosols and interphotoreceptor matrix were examined collectively for the presence of interstitial retinol-binding protein (IRBP). Although there was depletion of the amount of vitamin A stored in the RPE, this was commensurate with the histopathologic findings on the RPE extent and thickness. No evidence was found for an accumulation of free retinol. Nearly all of the vitamin A stored in the RPE was esterified. As in normal eyes, the retinyl esters consisted mainly of palmitate mixed with a small proportion of stearate. Eleven-cis retinyl esters were present, although their proportion was lower than that reported for normals. IRBP could not be detected in stained gels of the soluble proteins, or by autoradiography of these gels after treatment with 125I-concanavalin A. These findings suggest that depletion of stored vitamin A, accumulation of free retinol, or deficiency of 11-cis isomer are unlikely to be causative factors in the retinal degeneration examined here. Although the depletion of IRBP seen at this advanced stage might be secondary to the advanced loss of photoreceptors, the authors cannot rule out the possibility that a relative deficiency or abnormality in this protein at earlier disease stages may contribute to the pathogenesis of retinitis pigmentosa.

Retinoid-binding proteins and retinol esterification in cultured retinal pigment epithelium cells.

The esterification of all-trans retinol and the occurrence of cytosolic retinoid-binding proteins was investigated in cultured bovine retinal pigment epithelium (RPE) cells. (3)H-labeled all-trans retinyl ester (mainly palmitate) was formed at an initial rate of 0.1 nmol.mg protein(?1).min(?1) when (3)H-labeled all-trans retinol was incubated with the 100,000 g pellet obtained from a homogenate of freshly-harvested cells. No esterification could be detected under the same conditions after 14 days in culture in defined medium (DM) or in medium containing 20% fetal bovine serum (CM). No enhancement or restoration of esterifying capacity was observed when the assay mixture was supplemented with palmitoyl CoA. As determined by specific, saturable binding of (3)H-labeled all-trans retinol and (3)H-labeled 11-cis retinal to proteins with mol. wts 16,000 and 33,000 dalton on calibrated Bio-Sil TSK 250 size-exclusion columns, the cytosol of freshly-harvested RPE cells contained cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRAlBP). By comparison with the quantity of (3)H-labeled all-trans retinol bound under identical conditions to pure dog liver CRBP, it was estimated that fresh RPE cells contained 102 +/- 3 ng CRBP.?g cytosol protein(?1). In cultured and subcultured cells, CRBP was present at much lower levels (down to one-tenth of the initial amounts) and CRAlBP could not be detected. Since binding of (3)H-labeled all-trans retinoic acid to a protein with molecular weight of 17,000 dalton was not observed in the cytosols of fresh or cultured cells, it was concluded that cellular retinoic acid binding protein (CRABP) was either present at very low levels or absent altogether. An unidentified peak of specific (3)H-labeled all-trans-retinoic acid binding at mol. wt 61,000 dalton was prominent in subcultured cells. These results show that in RPE cells in culture the expression of differentiated phenotype with respect to retinoid utilization undergoes significant modification. It is postulated that changes in the composition of the extracellular matrix (e.g. absence of interstitial retinol-binding protein, IRBP) may be involved.

Utilization of exogenous retinol by frog pigment epithelium.

High-performance liquid chromatography was used to investigate the utilization of exogenous 11,12-3H2-retinol by frog pigment epithelium (RPE) in vitro or after intraocular injection into the dark-adapted, whole animal. Isolated frog RPE contains an adequate supply of acyl donors and can esterify all-trans, 11-cis and 13-cis isomers of retinol. The esterifying activity is restricted to the particulate fraction. Homogenates of choroid cannot esterify retinol. The ester formed by the RPE is primarily palmitate, and is therefore identical with the endogenous retinyl ester. Frog RPE also formed 13-cis retinyl palmitate from all-trans retinol, probably by esterification of 13-cis retinol formed non-enzymatically from the all-trans isomer. None of the in vitro experiments provided any evidence for the formation of 11-cis retinoid. There was slow appearance of label in 11-cis retinyl palmitate when 3H-all-trans retinol was injected intraocularly into the intact frog. After 15 hr its specific activity was only 20% of that of the all-trans retinyl palmitate. This rate of formation of 11-cis retinoid is inadequate for rhodopsin regeneration. However, it is more than an order of magnitude too fast to be accounted for by phagocytosis of rhodopsin. It is suggested that 11-cis retinoid is generated in the retina and is slowly transferred to the site of esterification in the RPE.

Vitamin A transport between retina and pigment epithelium--an interstitial protein carrying endogenous retinol (interstitial retinol-binding protein).

We have demonstrated and partially characterized an interstitial retinol-binding protein (IRBP) confined to bovine interphotoreceptor matrix (IPM). The native protein is a concanavalin A-binding glycoprotein with a mol. wt of 260 k as measured by gel-filtration and size-exclusion high-performance liquid chromatography. On SDS-gels, its mol. wt is 140-145 k. Since the protein is glycosylated, this value is probably too high. Hence, the native protein may be a dimer consisting of two identical subunits. The endogenous ligand has been analyzed by high-performance liquid chromatography--it consists mainly of all-trans retinol. Occasionally, retinal and 11-cis retinol are also associated with it. The amount of retinol bound to IRBP increases when the eyes are illuminated. The total binding capacity was estimated to represent 4-5% of the retinol released from a total rhodopsin bleach. We have established that, like serum retinol-binding protein, IRBP can be also bind retinoic acid, although it has not been established that retinoic acid is an endogenous ligand. The fluorescence emission lambda max for IRBP with its native ligand is at 470 nm and the excitation lambda max for this fluorescence is at 333 nm. Other retinoid carriers in the interphotoreceptor matrix have molecular weights of about 15 and 33 k. These probably correspond to cellular retinol- and retinal-binding proteins, respectively. Since both proteins have been identified in the pigment epithelium and retina cytosols, their presence in the IPM could be a result of cell damage. We conclude that interstitial retinol-binding protein is the best candidate for a transport protein carrying retinol between the rod outer segments and the pigment epithelium.

Use of high-performance liquid chromatography in the analysis of retinyl and 3,4-didehydroretinyl compounds in tissue extracts of bullfrog tadpoles and goldfish.

HPLC (high-performance liquid chromatography) was used to analyse retinyl and 3,4-didehydroretinyl compounds in tissue extracts from goldfish and bullfrog tadpoles. Using silica columns (packed with 10-micron mu Porasil or 5-micron Ultrasphere particles) eluted with n-hexane (containing a small amount of dioxane or diethyl ether), the authentic all-trans retinyl and 3,4-didehydroretinyl palmitates, retinal and 3,4-didehydroretinal, retinol and 3,4-didehydroretinol were completely separated. Liver and eye extracts of the goldfish and bullfrog tadpoles had mainly esterified all-trans retinol and all-trans 3,4-didehydroretinol. In the liver, these vitamin A were conjugated to a number of fatty acids whereas in the eye, principally one fatty acid was used. Moreover, the relative proportions of all-trans retinol and all-trans 3,4-didehydroretinol (obtained by analysing the saponified esters) were significantly different between some of these body compartments.

Visual cycle in the mammalian eye. Retinoid-binding proteins and the distribution of 11-cis retinoids.

This work was designed to provide an insight into the mammalian visual cycle by investigating the possible function of retinoid-binding proteins in this system, and the distribution and type of 11-cis retinoids present in the interphotoreceptor matrix and the cytosols of the retinal pigment epithelium and retina. The total retinol and retinal in the soluble fractions from these three compartments was 8% (3.31 nmol/eye) of the retinyl palmitate and stearate stored in the pigment epithelium membrane fractions (39 nmol/eye). Only small amounts of retinoids were detected in the rod outer segment cytosol. The insoluble fractions also contained retinol, nearly all of which was found in the retina. The retinoids in the soluble fractions appeared to be bound to cellular retinol-binding protein (CRBP), cellular retinal-binding protein (CRA1BP) and interstitial retinol-binding protein (IRBP, a high-Mr glycoprotein). Using immunospecific precipitation, immunoblot and immunocytochemical techniques it was demonstrated that IRBP was localized in the interphotoreceptor matrix and was synthesized and secreted by the retina, a process that did not require the protein to be glycosylated. The amount of retinol bound to IRBP increased if the eyes were exposed to light, when it was estimated that the protein carried up to 30% of its full capacity for all-trans retinol. In addition to all-trans retinol, IRBP carried smaller amounts of 11-cis retinol. The proportion of 11-cis retinol was frequently higher in eyes that had been protected from illumination, suggesting that IRBP plays a role in rhodopsin regeneration during dark-adaptation. Additionally, endogenous 11-cis retinoids in the retina and RPE cytosols were bound to an Mr 33,000 protein tentatively identified as CRA1BP. The 11-cis retinoid in the retina cytosol was mainly in the form of retinol, while in the RPE cytosol it was mainly in the form of retinal. Substantial amounts of 11-cis retinol were also found in the insoluble (membrane) fraction from the retina. It is suggested that in the mammalian retina 11-cis retinol is generated from all-trans retinol (possibly in the Muller cells). Lack of an 11-cis retinol oxidoreductase in the retina prevents it from being utilized for rhodopsin regeneration until it has been transported to the pigment epithelium, where it is converted to 11-cis retinal and returned to the rod outer segments. It is also suggested that IRBP may be implicated in the transport of retinoids between the rod outer segments, the Muller cells and the pigment epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Bovine interstitial retinol-binding protein (IRBP)--isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA.

Three clones for b-IRBP were isolated by anti b-IRBP screening of two bovine retina libraries in the expression vector lambda gt11. The cDNA inserts were then used as hybridization probes to screen and isolate three more clones in a bovine retina library in the non-expression vector lambda gt10. The six overlapping clones generated a b-IRBP cDNA sequence of 3400 nucleotides. An open reading frame encoded the complete amino acid sequences of 8 of the 35 b-IRBP tryptic peptides purified in the present study. One tentative glycosylation site was identified. The coding region was followed by TAG translation terminating codon and an untranslated stretch of about 1700 nucleotides that ended in a sequence containing a presumptive AATAAA polyadenylation signal that was 18 nucleotides upstream from a 10 nucleotide oligo(A) tract. The coding region for b-IRBP would be expected to be 3300 bp long, but Northern blot hybridization experiments performed with bovine retina polyadenylated RNA and probes containing part of the coding region established that the mRNA for b-IRBP consisted of a major species of about 6300 bp, and a minor species of 5200 bp. In vitro translation of bovine retina polyadenylated RNA in a rabbit reticulocyte lysate system yielded an immunoreactive protein that was comparable in size with nonglycosylated, mature IRBP, showing that it is not synthesized from a large precursor, and supporting our finding that the mRNA contains an extensive non-coding region.