Retinal Dehydrogenase Research Articles

Autoantigens in ovarian autoimmunity associated with unexplained infertility and premature ovarian failure

To identify ovarian autoantigens associated with ovarian autoantibodies. Hypothesis-generating prospective study. Urban infertility referral centers and academic research institution. Seventy-four patients with infertility, 19 patients with premature ovarian failure (POF), and 16 healthy control women. None. Identification of autoantigens. To identify major antigens for ovarian autoimmunity, sera from 74 women with unexplained infertility were screened for ovarian autoantibodies (AOAs) by immunoassay and one-dimensional Western blot. The majority of sera had immunoreactions at 50-56 kDa. Six representative positive infertility sera were used to identify antigens between 40 and 60 kD by two-dimensional Western blot and mass spectrometry. Antigens included aldehyde (retinal) dehydrogenases (ALDH1A1, ALDH1A2, and ALDH7A1), protein disulfide isomerase A3, vimentin, α-enolase, phosphoglycerate dehydrogenase, and selenium-binding protein 1 (SBP1). Sixty percent (24 out of 40) of infertility and POF sera were positive for recombinant ALDH1A1, SBP1, or enolase; 80.7% (21 out of 26) of AOA-positive sera had antibodies to one or more of the three antigens, and only 7% (1 out of 14) of AOA-negative sera had antibodies to recombinant proteins. ALDH1A1 and SBP1 are unique to ovarian autoimmunity associated with infertility and POF, and may provide the basis for specific tests to identify patients with ovarian autoimmunity.

Multiple retinol dehydrogenases (short‐chain dehydrogenases) and retinal dehydrogenases synthesize retinoic acid in hippocampus astrocytes

Little direct evidence identifies hippocampus cell types and enzymes that biosynthesize all‐trans retinoic acid (atRA). We show that primary rat astrocytes, but not neurons, biosynthesize atRA using retinol dehydrogenases (RDH) of the short‐chain dehydrogenase/reductase (SDR) gene family, and retinaldehyde dehydrogenases (RALDH). Astrocytes secrete atRA into their medium; neurons sequester atRA. Neurons and astrocytes both synthesize retinyl esters (RE). atRA autoregulates its concentrations by inducing RE synthesis (lecithin:retinol acyltransferase, LRAT) and stimulating its catabolism (cytochrome P450 CYP26B1). Knockdown by RNAi indicates that RDH10, RDH2, RALDH1, 2 and 3 contribute to atRA production. Knockdown of the SDR DHRS9 increases RA synthesis ~50% thru increasing RALDH1 expression. Culture‐time dependent expression of RALDH1 and 2, RDH10, DHRS9, LRAT and CYP26B1 correlate with atRA and RE synthesis, suggesting intrinsic adaption of retinoid homeostasis. These data show that adult hippocampus astrocytes rely on multiple RDHs and RALDHs to provide a paracrine source of atRA to neurons. Observation of cross talk between DHRS9 and RALDH1 provides novel insight into regulation of RA biosynthesis. Supported by NIH grant DK36870.

Human duodenal proteome modulations by glutamine and antioxidants

Glutamine (Gln) has protective, anti-inflammatory effects in animal models and humans. Antioxidant nutrients may exert synergistic effects on intestinal functions. Therefore, these combined nutrients may have a therapeutic potential during intestinal inflammation. This study was designed to investigate in humans the effects of a supplement composed of Gln and high-dosed antioxidant micronutrients compared to isomolar Gln only, on duodenal proteome. Enteral perfusion of Gln (0.8 mmol x kg(-1) x h(-1)) or supplement was performed in two groups of six healthy volunteers during 5 h before taking endoscopic duodenal biopsies. Protein expression was analyzed by 2-DE and the relevant proteins identified by MS/MS. About 1500 protein spots were revealed in both supplement and Gln conditions. Comparative proteomics analysis indicated that 11 proteins were differentially and significantly (p≤0.05) expressed in response to the supplement. These proteins were essentially implicated in metabolism pathways, e.g. fatty acid binding protein-1 and 40S ribosomal protein SA expressions were downregulated while manganese superoxide dismutase and retinal dehydrogenase-1 expressions were upregulated. This study provides new information on human duodenal proteome and its nutritional modulation, and supports further clinical investigations designed to evaluate the effects of Gln plus antioxidants during intestinal inflammation and cancer.

Kidneys of Alb/TGF-β1 Transgenic Mice Are Deficient in Retinoic Acid and Exogenous Retinoic Acid Shows Dose-Dependent Toxicity

Background: Alb/TGF-β₁ transgenic mice overexpress active transforming growth factor-β₁ (TGF-β₁) in the liver, leading to increased circulating levels of the cytokine and progressive renal fibrosis. This study was designed to explore if exogenous all-trans retinoic acid (tRA) prevents renal fibrosis in this animal model. Methods: The retinoid profile in kidney and liver of wild-type and Alb/TGF-β₁ transgenic mice was examined by high-performance liquid chromatography and slow-release pellets containing different amounts of tRA were implanted subcutaneously to treat the Alb/TGF-β₁ transgenic mice, starting at 1 week of age; mice were sacrificed 2 weeks later. Results: Kidneys of 3-week-old wild-type mice had abundant tRA, which was completely absent in kidneys of the transgenic mice. Low doses of tRA (6–10.7 mg/kg/day) failed to affect renal fibrosis although it tended to suppress the mRNA expression of some molecular markers of fibrosis and retinal dehydrogenase 2 (RALDH2), a gene encoding a key tRA-synthesising enzyme. These tendencies disappeared, mortality tended to increase and RALDH2 and connective tissue growth factor (CTGF) mRNAs significantly increased in the medium-dose group (12.7–18.8 mg/kg/day). High doses (20.1–27.4 mg/kg/day) showed even higher toxicity with increased renal fibrosis and significant mortality. Conclusions: Alb/TGF-β₁ transgenic mice are characterised by depletion of endogenous renal tRA. Exogenous tRA dose-dependently increases mortality and kidney fibrosis, which is associated with dose-dependent regulation of renal RALDH2 and CTGF mRNA expression.

Lymph Node Stromal Cells Support Dendritic Cell-Induced Gut-Homing of T Cells

T cells are imprinted to express tissue-specific homing receptors upon activation in tissue-draining lymph nodes, resulting in their migration to the site of Ag entry. Expression of gut-homing molecules alpha(4)beta(7) and CCR9 is induced by retinoic acid, a vitamin A metabolite produced by retinal dehydrogenases, which are specifically expressed in dendritic cells as well as stromal cells in mucosa-draining lymph nodes. In this study, we demonstrate that mesenteric lymph node stromal cell-derived retinoic acid can directly induce the expression of gut-homing molecules on proliferating T cells, a process strongly enhanced by bone marrow-derived dendritic cells in vitro. Therefore, cooperation of sessile lymph node stromal cells with mobile dendritic cells warrants the imprinting of tissue specific homing receptors on activated T cells.

Retinoic acid signalling induces the differentiation of mouse fetal liver‐derived hepatic progenitor cells

Hepatic progenitor cells (HPCs) can be isolated from fetal liver and extrahepatic tissues. Retinoic acid (RA) signalling plays an important role in development, although the role of RA signalling in liver-specific progenitors is poorly understood. We sought to determine the role of RA in regulating hepatic differentiation. RNA was isolated from liver tissues of various developmental stages. Liver marker expression was assessed by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Reversibly immortalized HPCs derived from mouse embryonic day 14.5 (E14.5) liver (aka, HP14.5) were established. Albumin promoter-driven reporter (Alb-GLuc) was used to monitor hepatic differentiation. Glycogen synthesis was assayed as a marker for terminal hepatic differentiation. Retinoic acid receptor (RAR)-alpha, retinoid X receptor (RXR)-alpha and RXR-gamma expressed in E12.5 to postnatal day 28 liver samples. Expression of RAR-beta and RXR-beta was low perinatally, whereas RAR-gamma was undetectable in prenatal tissues and increased postnatally. Retinal dehydrogenase 1 and 2 (Raldh1 and Raldh2) were expressed in all tissues, while Raldh3 was weakly expressed in prenatal samples but was readily detected postnatally. Nuclear receptor corepressors were highly expressed in all tissues, while expression of nuclear co-activators decreased in perinatal tissues and increased after birth. HP14.5 cells expressed high levels of early liver stem cell markers. Expression of RA signalling components and coregulators was readily detected in HP14.5. RA was shown to induce Alb-GLuc activity and late hepatocyte markers. RA was further shown to induce glycogen synthesis in HP14.5 cells, an important function of mature hepatocytes. Our results strongly suggest that RA signalling may play an important role in regulating hepatic differentiation.

Kinetic characterization of recombinant mouse retinal dehydrogenase types 3 and 4 for retinal substrates

Background Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis. Methods RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography. Results RALDH3 oxidized all- trans retinal with high catalytic efficiency ( V max/ K m = 77.9) but did not show activity for either 9- cis or 13- cis retinal substrates. On the other hand, RALDH4 was inactive for all- trans retinal substrate, exhibited high activity for 9- cis retinal oxidation ( V max/ K m = 27.4), and oxidized 13- cis retinal with lower catalytic efficiency ( V max/ K m = 8.24). β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9- cis and 13- cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl 2 activated 13- cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9- cis retinal oxidation, and slightly activated RALDH3 activity. Conclusions These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates. General significance The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.

Retinoic acid selectively inhibits death of basal vomeronasal neurons during late stage of neural circuit formation

In mouse, sexual, aggressive, and social behaviors are influenced by G protein-coupled vomeronasal receptor signaling in two distinct subsets of vomeronasal sensory neurons (VSNs): apical and basal VSNs. In addition, G protein-signaling by these receptors inhibits developmental death of VSNs. We show that cells of the vomeronasal nerve express the retinoic acid (RA) synthesizing enzyme retinal dehydrogenase 2. Analyses of transgenic mice with VSNs expressing a dominant-negative RA receptor indicate that basal VSNs differ from apical VSNs with regard to a transient wave of RA-regulated and caspase 3-mediated cell death during the first postnatal week. Analyses of G-protein subunit deficient mice indicate that RA and vomeronasal receptor signaling combine to regulate postnatal expression of Kirrel-2 (Kin of IRRE-like), a cell adhesion molecule regulating neural activity-dependent formation of precise axonal projections in the main olfactory system. Collectively, the results indicate a novel connection between pre-synaptic RA receptor signaling and neural activity-dependent events that together regulate neuronal survival and maintenance of synaptic contacts.

Divergent Roles for Airway Epithelial MMP7 and Retinoic Acid in Experimental Asthma (140.5)

Abstract The innate immune response of airway epithelial cells to aeroallergen likely initiates the development of T cell responses that are central to allergic inflammation. Although proteolytically active allergens induce the expression of interleukin (IL)-25/IL-17E, we show that epithelial matrix metalloproteinase 7 (MMP7) is expressed in human asthma, and is required for maximal activity of this cytokine in promoting T helper type 2 cell differentiation. Allergen-challenged MMP7-/- mice showed reduced airway hyperreactivity, allergic inflammatory cytokine, and increased expression of retinal dehydrogenase (RALDH)-1. Inhibition of RALDH-1 restored the asthma phenotype in MMP7-/- mice and inhibited lung T regulatory cell responses while exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating IL-25 while simultaneously inhibiting retinoid-dependent T regulatory cell development.

Divergent functions for airway epithelial matrix metalloproteinase 7 and retinoic acid in experimental asthma

The innate immune response of airway epithelial cells to aeroallergen initiates the development of T cell responses that are central to allergic inflammation. Although proteinase allergens induce the expression of interleukin 25 we show that epithelial matrix metalloproteinase 7 (MMP7) was expressed in asthma and was required for maximal activity of IL-25 in promoting T helper type 2 cell differentiation. Allergen-challenged Mmp7−/− mice showed reduced airway hyperreactivity, allergic inflammatory cytokine production and increased expression of retinal dehydrogenase (RALDH)-1. Inhibition of RALDH-1 restored the asthma phenotype in Mmp7−/− mice and inhibited lung T regulatory cell responses while exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating IL-25 while simultaneously inhibiting retinoid-dependent T regulatory cell development.

Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system

Retinoic acid (RA) is an important morphogen that regulates many biological processes, including the development of the central nervous system (CNS). Its synthesis from vitamin A (retinol) occurs in two steps, with the second reaction--catalyzed by retinal dehydrogenases (RALDHs)--long considered to be crucial for tissue-specific RA production in the embryo. We have recently identified the Xenopus homologue of retinol dehydrogenase 10 (XRDH10) that mediates the first step in RA synthesis from retinol to retinal. XRDH10 is specifically expressed in the dorsal blastopore lip and in other domains of the early embryo that partially overlap with XRALDH2 expression. We show that endogenous RA suppresses XRDH10 gene expression, suggesting negative-feedback regulation. In mRNA-injected Xenopus embryos, XRDH10 mimicked RA responses, influenced the gene expression of organizer markers, and synergized with XRALDH2 in posteriorizing the developing brain. Knockdown of XRDH10 and XRALDH2 by specific antisense morpholino oligonucleotides had the opposite effects on organizer gene expression, and caused a ventralized phenotype and anteriorization of the brain. These data indicate that the conversion of retinol into retinal is a developmentally controlled step involved in specification of the dorsoventral and anteroposterior body axes, as well as in pattern formation of the CNS. We suggest that the combinatorial gene expression and concerted action of XRDH10 and XRALDH2 constitute a ;biosynthetic enzyme code' for the establishment of a morphogen gradient in the embryo.

Metabolic memory puzzle and progression of diabetic retinopathy

Abstract Purpose Retinopathy is one of the most feared complications of diabetes. Good glycemic control can inhibit its development, but the effects of good glycemic control on the progression of retinopathy are not immediate. Diabetic patients may take years after re‐establishment of good glycemic control to show signs of arrest of its progression. Further, good glycemic control after a profound period of poor glycemic control does not immediately benefit the progression of retinopathy, and the imprinted effects of prior glycemic control produce the long lasting benefits of good glycemic control, thus suggesting a ‘metabolic memory’ phenomenon. Results Animal models of diabetic retinopathy, including dogs and rats, have duplicated this metabolic memory phenomenon. In rats, histopathology associated with diabetic retinopathy does not stop for at least six months when good glycemic control is initiated six months after induction of diabetes. Increase in retinal oxidative stress and peroxynitrite levels and activation of apoptosis execution enzyme‐caspase‐3 resist reversal after re‐institution of good glycemic control. Hyperglycemia‐induced inactivation of retinal glyceraldehyde dehydrogenase that is postulated to activate some of the key pathways associated with the development of diabetic complications remains inactive and covalently modified, and pro‐inflammatory markers elevated. Conclusion This suggests that the process of metabolic memory is complex, and multiple pathways contribute to this resistance of diabetic retinopathy to arrest. Understanding the mechanism responsible for the tendency of diabetic retinopathy to progress after reestablishment of good glycemic control should help reveal targets for therapies to prevent its progression.

Stromal Cells Confer Lymph Node-Specific Properties by Shaping a Unique Microenvironment Influencing Local Immune Responses

Lymph nodes (LN) consist not only of highly motile immune cells coming from the draining area or from the systemic circulation, but also of resident stromal cells building the backbone of the LN. These two cell types form a unique microenvironment which is important for initiating an optimal immune response. The present study asked how the unique microenvironment of the mesenteric lymph node (mLN) is influenced by highly motile cells and/or by the stromal cells. A transplantation model in rats and mice was established. After resecting the mLN, fragments of peripheral lymph node (pLN) or mLN were inserted into the mesentery. The pLN and mLN have LN-specific properties, resulting in differences of, for example, the CD103(+) dendritic cell subset, the adhesion molecule mucosal addressin cell adhesion molecule 1, the chemokine receptor CCR9, the cytokine IL-4, and the enzyme retinal dehydrogenase 2. This new model clearly showed that during regeneration stromal cells survived and immune cells were replaced. Surviving high endothelial venules retained their site-specific expression (mucosal addressin cell adhesion molecule 1). In addition, the low expression of retinal dehydrogenase 2 and CCR9 persisted in the transplanted pLN, suggesting that stromal cells influence the lymph node-specific properties. To examine the functional relevance of this different expression pattern in transplanted animals, an immune response against orally applied cholera toxin was initiated. The data showed that the IgA response against cholera toxin is significantly diminished in animals transplanted with pLN. This model documents that stromal cells of the LN are active players in shaping a unique microenvironment and influencing immune responses in the drained area.

Role of Matrix‐metalloprotease‐7 in a Model of Experimental Asthma

Bronchial asthma is a respiratory tract disease characterized by airway hyper‐responsiveness, mucus hyper‐production and inflammation. Airway epithelial cells are the initial sites where allergens are recognized. However, the role of epithelial cells in modulation of airway inflammation is not fully understood. One critical group of enzymes that regulate inflammatory processes in the lung is Matrix metalloproteases (MMP). We hypothesize that epithelial specific MMP7 modulates asthmatic phenotype by modifying inflammatory mediators produced by airway epithelium. We found that MMP7 null mice had reduced airway hyper‐reactivity in response to allergen challenge as well as fewer eosinophils in broncho‐alveolar lavage (BAL) fluid compared to WT mice. IL‐25 and Eotaxin, the critical mediators for asthma phenotype, were also reduced in MMP7−/− mice. Proteomic analysis of BAL fluid identified that Retinaldehydrogenase‐1 (RALDH‐1), a rate limiting epithelial specific enzyme for retinoic acid production, was up regulated in MMP7−/− asthmatic mice. In vivo inhibition of RALDH‐1, increased eotaxin protein expression in the airway and rescued the asthma phenotype in MMP7 −/− mice. Together, our data supports the role of MMP7 in modifying airway inflammation in asthma. We propose that identifying in vivo substrates for MMP7 may provide novel therapeutics for treatment of asthma.This work is funded by NIH grant (U19AI070973) to FK.

Retinoic acid

What is retinoic acid? Retinoic acid is a lipophilic molecule of low molecular weight (300 Da). Retinoic acid is synthesized from vitamin A (retinol) and found in embryos and adult vertebrates. Because animals are unable to produce vitamin A, they obtain this vitamin in the form of carotenoids from plants or retinyl esters derived from animal products. How is retinoic acid produced? In vertebrates, vitamin A, in the form of retinyl esters, is stored mainly in the liver as well as in the lungs, kidneys and bone marrow. Retinol, bound to the retinol-binding protein (RBP), enters the circulatory system in order to reach cells that require retinoic acid. The retinol–RBP complex enters the cells through the RBP receptor (STRA6). Within the cells, retinol is metabolized in a reversible step to retinal by the retinol or alcohol dehydrogenases (RODHs or ADHs) and then in an irreversible manner to retinoic acid by the retinal dehydrogenases (RALDHs). In the cytoplasm, retinoic acid is associated with the cellular retinoic acid-binding protein (CRABP). And what does it do? Simply speaking, retinoic acid functions to regulate gene expression (Figure 1). In cells, retinoic acid enters the nucleus where it binds to heterodimers formed by two different classes of nuclear receptors: Retinoic acid receptors (RARs) and retinoid X receptor (RXRs). The receptor heterodimers are bound to a specific DNA sequence known as the retinoic acid-response element (RARE) and upon retinoic acid binding, recruits co-activators (including histone acetyltransferase (HAT)), leading to transcription activation of specific retinoic acid-regulated genes. How many RARs and RXRs are there? In most vertebrates, there are three RARs (α, β and γ) and three RXRs (α, β and γ), all of which can have multiple isoforms, leading to large numbers of possible receptor combinations. How is the retinoic acid ‘signal’ turned off? In the cytoplasm, retinoic acid can be catabolized by the cytochrome P450 enzyme (CYP26) into inactive compounds that are then excreted. In the absence of retinoic acid, the RAR/RXR heterodimers bound to their target sequences recruit the nuclear receptor co-repressor complex NCOR/Sin3A/HDAC (histone deacetylase), inhibiting transcription activation. What does retinoic acid do? In adults, vitamin A and its retinoid derivatives play significant roles in a number of processes, such as vision, learning and memory, immune function, reproduction as well as the maintenance of epithelial function and differentiation. In the hippocampus, retinoic acid plays important physiological roles in synaptic plasticity, learning and memory, and adult neurogenesis. In the retina, retinoic acid acts as a light-signaling neuromodulator and regulates gap junction-mediated coupling of retinal neurons. It has also been implicated in the control of sleep and the circadian clock. Why are developmental biologists so obsessed with retinoic acid? This simple molecule has been implicated in the control of many aspects of embryo development that include cranio-facial morphogenesis, neuronal differentiation, eye, limb, lung, kidney and heart development. The patterning of the antero-posterior embryo body axis is also directly regulated by retinoic acid, which controls the expression of several HOX genes through RAREs in their regulatory/enhancer regions. Are there any human diseases related to retinoic acid deficiency? Reduction of vitamin A levels in the diet can lead to a debilitating immune system, anemia or blindness. During pregnancy, insufficient levels of vitamin A lead to fetal vitamin A deficiency-induced syndrome. This results in developmental defects exhibiting many common features with those observed in the RAR/RXR mutations in mouse, including cranio-facial and eye abnormalities. Retinoic acid as an anticancer drug…? Acute promyelocytic leukaemia is caused by a chromosomal translocation that fuses the promyelocytic leukaemia gene (PML) on chromosome 15 with the RARα gene on chromosome 17. The PML-RARα fusion protein leads to a recruitment of co-repressor complexes that epigenetically silence gene expression. Differentiation therapy with retinoic acid is being used in combination with chemotherapy for the treatment of patients with acute promyelocytic leukaemia, resulting in a 70-80% cure rate. Retinoic acidVilhais-Neto et al.Current BiologyApril 08, 2008In Brief(Current Biology 18, R191–R192, March 11, 2008) Full-Text PDF Open Archive

Triiodothyronine stimulates CMO1 gene expression in human intestinal Caco-2 BBe cells

Vitamin A is derived from provitamin A carotenoids, mainly β-carotene, by β-carotene 15,15′-monooxygenase (CMO1; EC 1.13.11.21). We previously found that enhancement of CMO1 mRNA expression was related to the levels of hormones, such as thyroid hormones, in chick duodenum. We investigated whether CMO1 expression was increased by triiodothyronine (T 3), a thyroid hormone, using human intestinal Caco-2 BBe cells. Treatment of 7 days post-confluent Caco-2 BBe cells with T 3 significantly enhanced CMO1 mRNA levels in both dose- and time-dependent manners. This T 3-inducing effect on CMO1 mRNA level was blocked by actinomycin D. The levels of mRNAs for the thyroid hormone receptors TRα1 and TRβ1 were significantly increased in 7 days post-confluent Caco-2 BBe cells. CMO1 enzyme activity was also significantly increased by T 3 treatment in medium supplemented with fetal bovine serum. Furthermore, T 3 treatment also increased the level of mRNA for lecithin:retinol acyltransferase (LRAT), but not those for cellular retinol-binding protein, type II (CRBPII) and retinal dehydrogenase 1 (RALDH1), in Caco-2 BBe cells. These results indicate that T 3 is an important hormone for the regulation of vitamin A and β-carotene metabolism-related gene expression in human small intestinal cells.

Expressions of Raldh3 and Raldh4 during zebrafish early development

Retinoic acid (RA) plays crucial roles in vertebrate embryogenesis. Four retinal dehydrogenases (Raldhs) that are responsible for RA synthesis have been characterized in mammals. However, only Raldh2 ortholog is identified in zebrafish. Here, we report the identification of raldh3 and raldh4 genes in zebrafish. The predicted proteins encoded by zebrafish raldh3 and raldh4 exhibit 70.0% and 73.5% amino acid identities with mouse Raldh3 and Raldh4, respectively. RT-PCR analyses reveal that both raldh3 and raldh4 mRNAs are present in early development. However, whole mount in situ hybridization shows that raldh3 mRNA is first expressed in the developing eye region of zebrafish embryos at 10-somite stage. At 24 hpf (hours post fertilization), raldh3 mRNA is expressed in the ventral retina of eye. At 36 hpf, the mRNA is also expressed in otic vesicle in addition to ventral retina, and it maintains its expression pattern till 2 dpf (days post fertilization). At 3 dpf, raldh3 mRNA becomes very weak in ventral retina but is present in otic vesicle at a high level. At 5 dpf and 7 dpf, raldh3 is no longer expressed in eyes but is expressed in otic vesicles at a very low level. raldh4 mRNA is initially detected in developing liver and intestine regions at 2 dpf embryos. Its expression level becomes very high in these two regions of embryos from 3 dpf to 5 dpf. Analysis on the sections of 5 dpf embryos reveals that raldh4 is expressed in the epithelium of intestine. At 7 dpf, raldh4 mRNA is only weakly expressed in the epithelium of intestinal bulb.

Direct analysis of retinal dehydrogenase activity on an electroblotting membrane following separation by non-denaturing two-dimensional electrophoresis

The reaction from retinal to retinoic acid catalyzed by retinal dehydrogenase on a polyvinylidene difluoride (PVDF) membrane was examined using laser desorption ionization time of flight mass spectrometry (LDI-TOF MS) when the enzyme was separated by non-denaturing two-dimensional electrophoresis (2-DE), transferred onto the membrane, and stained without impairing the enzyme activity. Furthermore, the enzyme was analyzed by de novo sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after proteins from mouse liver were separated by non-denaturing 2-DE, blotted onto the membrane, and stained. The results indicated that the reported methods could be applied for the direct examination of changes in retinoid catalyzed by enzymes on such membranes.

In Vitro Induction of Mucosa-Type Dendritic Cells by All-Trans Retinoic Acid

Efficient induction of mucosal immunity usually employs nasal or oral vaccination while parenteral immunization generally is ineffective at generating mucosal immune responses. This relates to the unique ability of resident mucosal dendritic cells (DC) to induce IgA switching and to imprint mucosa-specific homing receptors on lymphocytes. Based on the well-established plasticity of the DC system, this study sought to investigate whether peripheral DC could be modulated toward "mucosa-type" DC by treatment with immunomodulatory, and therefore potentially adjuvant-like, factors. In this study, we show that monocyte-derived DCs pretreated with the vitamin A derivative all-trans retinoic acid (RA) indeed acquired several attributes characteristic of mucosal DC: secretion of TGF-beta and IL-6 and the capacity to augment mucosal homing receptor expression and IgA responses in cocultured lymphocytes. Addition of a TGF-beta-neutralizing Ab to cocultures significantly inhibited alpha4beta7 integrin, but not CCR9 mRNA expression by the lymphocytes. Both alpha4beta7 integrin and CCR9 mRNA expression, but not IgA production, were suppressed in the presence of a RA receptor antagonist. None of the observed effects on the lymphocytes were influenced by citral, a retinal dehydrogenase inhibitor, arguing against a role for de novo-synthesized RA. Collectively, our findings identified a novel role for RA as a mucosal immune modulator targeting DC. Our results further demonstrate that DC can act as efficient carriers of RA at least in vitro. Consequently, RA targeting of DC shows potential for promoting vaccine-induced mucosal immune responses via a parenteral route of immunization.

Developmental changes in gene expressions of β-carotene cleavage enzyme and retinoic acid synthesizing enzymes in the chick duodenum

Vitamin A is derived from provitamin A carotenoids, mainly β-carotene, by β-carotene 15,15′-monooxygenase (BCMO1; EC 1.13.11.21). We previously reported that chick duodenal BCMO1 activity increased abruptly just after hatching. In this study, we further investigated mechanisms and physiological roles of the postnatal induction of BCMO1 expression in the chick duodenum. We showed that BCMO1 mRNA levels increased in the chick duodenum during postnatal period after hatching, but remain unchanged in the chick liver throughout the perinatal period. Serum hydrocortisone (HC) levels were also increased after hatching. Moreover, HC-administered chicks showed an enhancement of duodenal BCMO1 mRNA during the perinatal period. We further analyzed the developmental gene expression patterns of three types of retinoic acid (RA) synthesizing enzymes in the chick duodenum. Among them, retinal dehydrogenase 1 (RALDH1) mRNA levels in the chick duodenum increased during the postnatal period, indicating a similar developmental expression pattern to that of BCMO1. These results suggest that the postnatal induction of BCMO1 gene expression in the chick duodenum may be caused by the elevation of serum HC levels and may contribute to the RALDH1-mediated RA synthetic pathway.