Genetic dissection in mice reveals a dynamic crosstalk between the delivery pathways of vitamin A.

Vitamin A is distributed within the body to support chromophore synthesis in the eyes and retinoid signaling in most other tissues. Two pathways exist for the delivery of vitamin A: the extrinsic pathway transports dietary vitamin A in lipoproteins from intestinal enterocytes to tissues, while the intrinsic pathway distributes vitamin A from hepatic stores bound to serum retinol binding protein (RBP). Previously, the intestine-specific homeodomain transcription factor (ISX) and the RBP receptor STRA6 were identified as gatekeepers of these pathways; however, it is not clear how mutations in the corresponding genes affect retinoid homeostasis. Here, we used a genetic dissection approach in mice to examine the contributions of these proteins in select tissues. We observed that ISX deficiency increased utilization of both preformed and provitamin A. We found that increased storage of retinoids in peripheral tissues of ISX-deficient mice was dependent on STRA6 and induced by retinoid signaling. In addition, double-mutant mice exhibited a partial rescue of the Stra6 mutant ocular phenotype. This rescue came at the expense of a massive accumulation of vitamin A in other tissues, demonstrating that vitamin A is randomly distributed when present in excessive amounts. Remarkably, provitamin A supplementation of mutant mice induced the expression of the RBP receptor 2 in the liver and was accompanied by increased hepatic retinyl ester stores. Taken together, these findings indicate dynamic crosstalk between the delivery pathways for this essential nutrient and suggest that hepatic reuptake of vitamin A takes place when excessive amounts circulate in the blood.

Identification of MFRP and the secreted serine proteases PRSS56 and ADAMTS19 as part of a molecular network involved in ocular growth regulation.

Precise regulation of ocular size is a critical determinant of normal visual acuity. Although it is generally accepted that ocular growth relies on a cascade of signaling events transmitted from the retina to the sclera, the factors and mechanism(s) involved are poorly understood. Recent studies have highlighted the importance of the retinal secreted serine protease PRSS56 and transmembrane glycoprotein MFRP, a factor predominantly expressed in the retinal pigment epithelium (RPE), in ocular size determination. Mutations in PRSS56 and MFRP constitute a major cause of nanophthalmos, a condition characterized by severe reduction in ocular axial length/extreme hyperopia. Interestingly, common variants of these genes have been implicated in myopia, a condition associated with ocular elongation. Consistent with these findings, mice with loss of function mutation in PRSS56 or MFRP exhibit a reduction in ocular axial length. However, the molecular network and cellular processes involved in PRSS56- and MFRP-mediated ocular axial growth remain elusive. Here, we show that Adamts19 expression is significantly upregulated in the retina of mice lacking either Prss56 or Mfrp. Importantly, using genetic mouse models, we demonstrate that while ADAMTS19 is not required for ocular growth during normal development, its inactivation exacerbates ocular axial length reduction in Prss56 and Mfrp mutant mice. These results suggest that the upregulation of retinal Adamts19 is part of an adaptive molecular response to counteract impaired ocular growth. Using a complementary genetic approach, we show that loss of PRSS56 or MFRP function prevents excessive ocular axial growth in a mouse model of early-onset myopia caused by a null mutation in Irbp, thus, demonstrating that PRSS56 and MFRP are also required for pathological ocular elongation. Collectively, our findings provide new insights into the molecular network involved in ocular axial growth and support a role for molecular crosstalk between the retina and RPE involved in refractive development.

Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae.

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.

Metabolic interactions between vitamin A and conjugated linoleic acid.

Lipid-soluble molecules share several aspects of their physiology due to their common adaptations to a hydrophilic environment, and may interact to regulate their action in a tissue-specific manner. Dietary conjugated linoleic acid (CLA) is a fatty acid with a conjugated diene structure that is found in low concentrations in ruminant products and available as a nutritional supplement. CLA has been shown to increase tissue levels of retinol (vitamin A alcohol) and its sole specific circulating carrier protein retinol-binding protein (RBP or RBP4). However, the precise mechanism of this action has not been elucidated yet. Here, we provide a summary of the current knowledge in this specific area of research and speculate that retinol and CLA may compete for catabolic pathways modulated by the activity of PPAR-α and RXR heterodimer. We also present preliminary data that may position PPAR-α at the crossroads between the metabolism of lipids and vitamin A.

Mimicry epitope from Ehrlichia canis for interphotoreceptor retinoid-binding protein 201-216 prevents autoimmune uveoretinitis by acting as altered peptide ligand.

We report here identification of novel mimicry epitopes for interphotoreceptor retinoid-binding protein (IRBP) 201-216, a candidate ocular antigen that causes experimental autoimmune uveoretinitis (EAU) in A/J mice. One mimicry epitope from Ehrlichia canis (EHC), designated EHC 44-59, induced cross-reactive T cells for IRBP 201-216 capable of producing T helper (Th)1 and Th17 cytokines, but failed to induce EAU in A/J mice. In addition, animals first primed with suboptimal doses of IRBP 201-216 and subsequently immunized with EHC 44-59 did not develop EAU; rather, the mimicry epitope prevented the disease induced by IRBP 201-216. However, alteration in the composition of EHC 44-59 by substituting alanine with valine at position 49, similar to the composition of IRBP 201-216, enabled the mimicry epitope to acquire uveitogenicity. The data provide new insights as to how microbes containing mimicry sequences for retinal antigens can prevent ocular inflammation by acting as naturally occurring altered peptide ligands.

Vitamin A status after prophylactic intramuscular vitamin A supplementation in extremely low birth weight infants.

BACKGROUND: Vitamin A supplementation (VAS) is recommended to prevent bronchopulmonary dysplasia (BPD). Our objective was to evaluate the effect of VAS on vitamin A (VA) status. We hypothesized that VAS would improve VA status in extremely low birth weight (ELBW) infants. MATERIALS AND METHODS: Retrospective chart review of infants 1 year before and after initiation of VAS (5000 IU 3 times a week intramuscularly [IM]; total 12 doses). Linear regression was used to model impact of VAS on VA status (retinol level and retinol/retinol binding protein [RBP] ratio). Models were adjusted for time and generalized estimating equations were used to account for intraindividual correlation. RESULTS: Sixty-seven infants (mean gestational age 26 ± 2 weeks; mean body weight 803 ± 142 g) were included; 35 received VAS and 32 did not (no-VAS). Both groups had similar baseline characteristics. Infants who received VAS had mean retinol levels that were 9.0 mcg/dL (95% confidence interval [CI], 4.9-13.2; P < .001) higher and mean retinol/RBP ratios that were 0.21 (95% CI, 0.07-0.36; P = .005) higher than the no-VAS group. Retinol and retinol/RBP ratio increased with time (P < .001). Fewer infants in the VAS group had VA deficiency (retinol/RBP ratios <0.7) compared with the no-VAS group. Culture-positive sepsis was more common in the VAS group (48% vs 12%; P = .002). CONCLUSIONS: VA status in ELBW infants was improved and maintained over the first month of life with IM VAS. Because of concerns for potential risks of repeated injections, further studies are indicated to evaluate the optimal mode of VA delivery in preterm infants.

Uptake of dietary retinoids at the maternal-fetal barrier: in vivo evidence for the role of lipoprotein lipase and alternative pathways.

Dietary retinoids (vitamin A and its derivatives) contribute to normal embryonic development. However, the mechanism(s) involved in the transfer of recently ingested vitamin A from mother to embryo is not fully understood. We investigated in vivo whether lipoprotein lipase (LPL) facilitates the placental uptake of dietary retinyl ester incorporated in chylomicrons and their remnants and its transfer to the embryo. We examined the effects of both genetic ablation (MCK-L0 mice) and pharmacological inhibition (P-407) of LPL by maintaining wild type and MCK-L0 mice on diets with different vitamin A content or administering them an oral gavage dose of [(3)H]retinol with or without P-407 treatment. We showed that LPL expressed in placenta facilitates uptake of retinoids by this organ and their transfer to the embryo, mainly through its catalytic activity. In addition, through its "bridging function," LPL can mediate the acquisition of nascent chylomicrons by the placenta, although less efficiently. Quantitative real-time PCR and Western blot analysis showed that placental LPL acts in concert with LDL receptor and LRP1. Finally, by knocking out the retinol-binding protein (RBP) gene in the MCK-L0 background (MCK-L0-RBP(-/-) mice) we demonstrated that the placenta acquires dietary retinoids also via the maternal circulating RBP-retinol complex. RBP expressed in the placenta facilitate the transfer of postprandial retinoids across the placental layers toward the embryo.

The interaction between retinol-binding protein and transthyretin analyzed by fluorescence anisotropy.

The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. Due to the tight binding of retinol within a substantially apolar binding site, an intense fluorescence emission characterizes the RBP-bound vitamin. The addition of TTR to the retinol-RBP complex (holoRBP) causes a marked increase in the fluorescence anisotropy of the RBP-bound retinol within the system, due to the formation of the holoRBP-TTR complex, which allows the interaction between the two proteins to be monitored. The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.

Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye.

In a previous study, we used differential proteomics to identify retinal proteins whose steady-state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady-state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina-retinal recycling and generation of retinoic acid-we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11-cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology.

Hepatic retinol secretion and storage are altered by dietary CLA: common and distinct actions of CLA c9,t11 and t10,c12 isomers.

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid obtained from ruminant products. Previous studies in rats and pigs showed that a dietary equimolar mixture of c9,t11 and t10,c12 CLA isomers induces changes in serum and tissue levels of retinoids (vitamin A derivatives). However, the mechanism(s) responsible for these actions remain(s) unexplored. Given the numerous crucial biological functions regulated by retinoids, it is key to establish whether the perturbations in retinoid metabolism induced by dietary CLA mediate some of the beneficial effects associated with intake of this fatty acid or, rather, have adverse consequences on health. To address this important biological question, we began to explore the mechanisms through which dietary CLA alters retinoid metabolism. By using enriched preparations of CLA c9,t11 or CLA t10,c12, we uncoupled the effects of these two CLA isomers on retinoid metabolism. Specifically, we show that both isomers induce hepatic retinyl ester accumulation. However, only CLA t10,c12 enhances hepatic retinol secretion, resulting in increased serum levels of retinol and its specific carrier, retinol-binding protein (RBP). Dietary CLA t10,c12 also redistributes retinoids from the hepatic stores toward the adipose tissue and possibly stimulates hepatic retinoid oxidation. Using mice lacking RBP, we also demonstrate that this key protein in retinoid metabolism mediates hepatic retinol secretion and its redistribution toward fat tissue induced by CLA t10,c12 supplementation.

Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.

BACKGROUND: Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling. RESULTS: The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each approximately 300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 microM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites. However, homology modeling of modules 1, 3 and 4 indicate that both sites may not be available for binding of ligands in all four modules. CONCLUSION: Although its four modules are homologous and each capable of supporting ligand-binding activity, structural differences between their ligand-binding domains, and interactions between the modules themselves will be critical to understanding IRBP's complex role in the visual cycle.

Effect of systemic progesterone concentration on the expression of progesterone-responsive genes in the bovine endometrium during the early luteal phase.

Increasing evidence indicates an association between the concentration of systemic progesterone during the early luteal phase of the oestrous cycle and embryo survival rate in cattle. We examined the relationship between the concentration of systemic progesterone on Days 4 to 8 post-ovulation and expression of progesterone receptor (PGR), oestrogen receptor +/- (ESR1) and retinol-binding protein (RBP) mRNA in the bovine endometrium. Heifers were blood sampled from the day of ovulation (Day 0) to Day 8 post-ovulation. On Day 4, animals were divided into low progesterone control (LC) and high progesterone control (HC) groups based on their plasma progesterone concentrations. Half of each group was supplemented with exogenous progesterone resulting in two further groups, low progesterone supplemented (LS) and high progesterone supplemented (HS). Endometrial tissues were recovered from all groups on Day 6 or Day 8 and gene expression was analysed following Northern blotting. Increasing progesterone concentrations were associated with decreased PGR and ESR1 expression. Duration-dependent effects of progesterone supplementation on ESR1 were evident and there was an effect of systemic progesterone concentrations between Day 0 and Day 4 on the expression of RBP at Days 6 and 8. Such progesterone-responsive changes in uterine gene expression are likely to affect embryo development.

Endogenous IRBP can be dispensable for generation of natural CD4+CD25+ regulatory T cells that protect from IRBP-induced retinal autoimmunity.

Susceptibility to experimental autoimmune uveitis (EAU), a model for human uveitis induced in mice with the retinal antigen interphotoreceptor retinoid-binding protein (IRBP), is controlled by "natural" CD4+CD25+ regulatory T (T reg) cells. To examine whether endogenous expression of IRBP is necessary to generate these T reg cells, we studied responses of IRBP knockout (KO) versus wild-type (WT) mice. Unexpectedly, not only WT but also IRBP KO mice immunized with a uveitogenic regimen of IRBP in complete Freund's adjuvant (CFA) exhibited CD25+ regulatory cells that could be depleted by PC61 treatment, which suppressed development of uveitogenic effector T cells and decreased immunological responses to IRBP. These EAU-relevant T reg cells were not IRBP specific, as their activity was not present in IRBP KO mice immunized with IRBP in incomplete Freund's adjuvant (IFA), lacking mycobacteria (whereas the same mice exhibited normal T reg cell activity to retinal arrestin in IFA). We propose that mycobacterial components in CFA activate T reg cells of other specificities to inhibit generation of IRBP-specific effector T cells in a bystander fashion, indicating that effective T reg cells can be antigen nonspecific. Our data also provide the first evidence that generation of specific T reg cells to a native autoantigen in a mouse with a diverse T cell repertoire requires a cognate interaction.

Integrating protein structures and precomputed genealogies in the Magnum database: examples with cellular retinoid binding proteins.

BACKGROUND: When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. RESULTS: The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1) multiple sequence alignments, 2) mapping of alignment sites to crystal structure sites, 3) phylogenetic trees, 4) inferred ancestral sequences at internal tree nodes, and 5) amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. CONCLUSION: We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural bioinformatics resources that are useful for identifying experimentally testable hypotheses about the molecular basis of protein behaviors and functions, as illustrated with the examples from the cellular retinoid binding proteins.

Identification of eight genes that are potentially involved in tamoxifen sensitivity in breast cancer cells.

Although the antiestrogen agent tamoxifen has long been used to treat women with hormone receptor positive invasive breast carcinoma, the mechanisms of its action and acquired resistance to tamoxifen during treatment are largely unknown. A number of studies have revealed that over-activation of some signaling pathways can cause tamoxifen resistance; however, very little information is available regarding the genes whose loss-of-function alternation contribute to tamoxifen resistance. Here we used a forward genetic approach in vitro to generate tamoxifen resistant cells from the tamoxifen sensitive breast cancer cell line ZR-75-1, and further identified the disrupted gene in different tamoxifen resistant clones. Retinol binding protein 7, DNA polymerase-transactivated protein 3, g-glutamyltransferase-like activity 1, slit-robo RhoGTPase-activating protein, tetraspan NET-4, HSPC194, amiloride-sensitive epithelial sodium channel gene, and Notch2, were the eight mutated genes identified in different tamoxifen resistant clones, suggesting their requirement for tamoxifen sensitivity in ZR-75-1 cells. Since the functions of these genes are not related to each other, it suggests that multiple pathways can influence tamoxifen sensitivity in breast cancer cells.

Thymic expression of peripheral tissue antigens in humans: a remarkable variability among individuals.

The majority of maturing T lymphocytes that recognize self-antigens is eliminated in the thymus upon exposure to their target antigens. This physiological process of negative selection requires that tissue-specific antigens be expressed by thymic cells, a phenomenon that has been well studied in experimental animals. Here, we have examined the expression in human thymi of four retinal antigens, that are capable of inducing autoimmune ocular disease retinal S-antigen (S-Ag), recoverin, RPE65 and inter-photoreceptor retinoid-binding protein (IRBP)], as well as four melanocyte-specific antigens, two of which are used as targets for melanoma immunotherapy [gp100, melanoma antigen recognized by T cells 1, tyrosinase-related protein (TRP)-1 and TRP-2]. Using reverse transcription (RT)-PCR, we found that all thymic samples from the 18 donors expressed mRNA transcripts of most or all the eight tested tissue antigens. Yet, the expression of the transcripts varied remarkably among the individual thymic samples. In addition, S-Ag, RPE65 and IRBP were detected by immunostaining in rare cells in sections of human thymi by antibodies against these proteins. Quantitative real-time RT-PCR analysis revealed that the retinal antigen transcripts in the human thymus are present at trace levels, that are lower by approximately five orders of magnitude than those in the retina. Our observations thus support the notions that thymic expression is a common feature for all tissue-specific antigens and that the levels of expression play a role in determining the susceptibility to autoimmunity against these molecules.

Pretreatment of starved rats with ornithine alpha-ketoglutarate: effects on hepatic mRNA levels and plasma concentrations of three liver-secreted proteins.

OBJECTIVE: Ornithine alpha-ketoglutarate (OKG) displays anabolic properties at the hepatic level, but the mechanisms involved remain unclear. This study investigated in vivo the ability of OKG to modulate hepatic gene expression of three liver-secreted proteins: albumin, transthyretin, and retinol binding protein. METHODS: One hundred eighty rats were fed for 5 d with a balanced regimen enriched with OKG (5 g.kg(-1).d(-1)) or an isonitrogenous mixture (alanine, glycine, and serine). Hepatic mRNA levels and plasma concentrations of the three proteins studied were determined at the end of the nutrition period and after 1, 2, and 3 d of food deprivation. Results were compared by analysis of variance and Bonferroni-Dunn tests. RESULTS: At the end of the nutrition period, hepatic mRNA levels and plasma concentrations of the three proteins were not modified by OKG supplementation. However, OKG largely increased mRNA levels of albumin, transthyretin, and retinol binding protein on the first day of starvation compared with control animals (+68%, +64% and +51%, respectively; P < 0.01 versus control). OKG precociously increased albuminemia (on day 2) but had no effect on plasma concentrations of transthyretin and retinol binding protein. Neither regulation of polyamine hepatic concentration nor alteration in hepatic amino acid content seemed to be implicated in these actions. CONCLUSION: This study is the first to demonstrate that OKG regulates in vivo liver gene expression during acute malnutrition by modulating hepatic mRNA levels.

Retinol-binding protein gene is highly expressed in higher-order association areas of the primate neocortex.

The neocortex consists of histochemically, connectionally, and functionally distinguishable areas. Recently, molecular biological techniques have enabled us to find rare types of genes expressed in specific neocortical areas. We previously reported occ1 gene as preferentially expressed in the primary visual cortex (V1), using the differential display method. Here, by differential display, we found selective and strong expression of the serum retinol-binding protein (RBP) gene, in higher-order association areas. In V1, RBP mRNA was expressed only in the superficial part of layer II, but its expression increased, involving deeper layers, along the visual pathway. In visual association areas such as TE, RBP mRNA was strongly expressed in both supra- and infragranular layers. In primary auditory and somatosensory areas, as in V1, RBP expression was low, and restricted to the upper part of the supragranular layers. The laminar pattern of RBP expression is in marked contrast with that of occ1; and in early visual areas where both genes are expressed, these occur in distinct sublayers within the supragranular layers. In neonatal monkeys, the area-specific expression pattern of RBP was less distinct, suggesting that the characteristic expression of RBP in higher-order association areas is mainly established postnatally.

Cellular retinol-binding protein-1 expression in endometrial stromal cells: physiopathological and diagnostic implications.

AIMS: Cellular retinol-binding protein-1 (CRBP-1) contributes to the maintenance of the differentiated state of the endometrium through retinol bioavailability regulation. The aim was to analyse CRBP-1 expression in endometrial stromal cells at eutopic and ectopic sites in different physiopathological conditions. METHODS AND RESULTS: Antibodies to CRBP-1, CD10 and alpha-smooth muscle actin were applied to proliferative (n = 10), secretory (n = 9) and atrophic (n = 7) endometrium, decidua (n = 4), adenomyosis (n = 5), endometriosis (n = 10), endometrial polyps (n = 9), simple endometrial hyperplasia (n = 6), well-differentiated endometrioid carcinoma (n = 6) and submucosal leiomyomas (n = 5). In some cases, Western blotting and reverse transcription-polymerase chain reaction were also applied. CRBP-1 was expressed by eutopic and ectopic endometrial stromal cells more markedly during the late secretory phase and in decidua of pregnancy. CRBP-1 expression was low in the stroma of atrophic endometrium and absent in myometrium, leiomyomas and cervical stroma. CD10 immunoreactivity was weak in atrophic endometrium and in decidua. CONCLUSIONS: CRBP-1 expression characterizes endometrial stromal cells at eutopic and ectopic sites and appears to be more specific than CD10. The level of CRBP-1 varies in intensity according to hormonal variations, reaching its maximum in predecidua and decidua. Thus, immunodetection of CRBP-1 may help to elucidate the physiopathological changes which occur in endometrial stroma and can also be applied as an adjuvant stromal marker.

Role of purpurin as a retinol-binding protein in goldfish retina during the early stage of optic nerve regeneration: its priming action on neurite outgrowth.

Unlike mammals, the fish optic nerve can regenerate after injury. So far, many growth or trophic factors have been shown as an axon-regenerating molecule. However, it is totally unknown what substance regulates or triggers the activity of these factors on axonal elongation. Therefore, we constructed a goldfish retina cDNA library prepared from the retina treated with optic nerve transection 5 d previously, when it was just before regrowing optic axons after injury. A cDNA clone for goldfish purpurin for which expression was upregulated during the early stage of optic nerve regeneration was isolated from the retina cDNA library. Purpurin was discovered as a secretory retinol-binding protein in developing chicken retinas. Levels of purpurin mRNA and protein transiently increased and rapidly decreased 2-5 d and 10 d after axotomy, respectively. Purpurin mRNA was localized to the photoreceptor cells, whereas the protein was diffusely found in all of the retinal layers. A recombinant purpurin alone did not affect any change of neurite outgrowth in explant culture of the control retina, whereas a concomitant addition of the recombinant purpurin and retinol first induced a drastic enhancement of neurite outgrowth. Furthermore, the action of retinol-bound purpurin was effective only in the control (untreated) retinas but not in those primed (treated) with a previous optic nerve transection. Thus, purpurin with retinol is the first candidate molecule of priming neurite outgrowth in the early stage of optic nerve regeneration in fish.