Retinoid Binding Research Articles

Analysis of Ligand Binding and Protein Dynamics of Human Retinoid X Receptor Alpha Ligand-Binding Domain by Nuclear Magnetic Resonance

Retinoid X receptors (RXRs) are nuclear receptors that can activate transcription as homodimers or as obligate heterodimeric partners of other nuclear receptors. While the crystal structures of the RXR ligand-binding domains (LBD) have been previously determined, the dynamics of activation is less well characterized at an atomic level. To probe the effect of ligand binding on RXR LBD dynamics, we initiated nuclear magnetic resonance studies of recombinant human RXRalpha LBD (T223-T462) with and without bound 9-cis-retinoic acid (9cRA). The 1HN, 15N, 13C(alpha), 13CO, and 13C(beta) resonance assignments were established for 164 of 240 residues in apo-RXRalpha LBD. Resonances corresponding to an additional 47 residues emerged upon 9cRA binding. These additional residues included those located in the vicinity of the ligand-binding pocket (helices H3, H5, and strands S1, S2), as well as residues located at the dimerization interface (helices H9 and H10). Thus 9cRA binding stabilized the ligand-binding pocket and had allosteric effects on the dimerization interface. Ligand-induced chemical shift perturbations outside the binding cavity were mapped to helix H3 and the AF-2 helix H12, indicating conformational changes in these regions. However, helix H11, a component of the tetramerization interface, and a large part of helix H10, a component of the dimerization interface, remained undetectable even after 9cRA binding. Although apo- and holo-hRXRalpha LBD existed predominantly as homodimers in solution, exchange between monomeric, dimeric, and tetrameric forms of the protein could have contributed to line broadening of cross-peaks corresponding to helices H10 and H11. 15N T1, T2, and steady-state {1H}-15N NOE data collected at 500 and 700 MHz static magnetic fields showed that the internal motions for the residues in the H1-H3 loop (K245-D263) were much less restricted than those in the protein core for both apo- and holo-forms. Significant exchange R(ex) contributions to the transverse relaxation rate were detected for most of the residues measured in both apo- and holo-RXRalpha LBDs by transverse relaxation optimized spectroscopy-Carr-Purcell-Meiboom-Gill (CPMG) experiments at two B1 field strengths. Taken together these results suggest that the RXRalpha LBD exists as a dynamic ensemble of conformations, even after binding its cognate ligand. Such dynamic characteristics may allow RXRalpha to partner with multiple nuclear receptors.

Defining the Retinoid Binding Site in the Rod Cyclic Nucleotide-gated Channel

Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-trans-retinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521–531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, β-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid–channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues ∼15 Å away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.

Specificity of Binding of all-trans-Retinyl Ester to RPE65

Membrane-bound RPE65 (mRPE65) is a binding protein for all-trans-retinyl esters, which are the substrates for the isomerization reaction that completes the visual cycle. RPE65 is essential for rhodopsin regeneration and, hence, for vision. As RPE65 appears to be part of the rate-limiting pathway in the visual cycle, specific antagonists of the molecule will be important in evaluating its full physiological role. The protein is known to stereoselectively bind all-trans-retinyl esters (tREs), with dissociation constants in the 50 nM range. This study explores the overall binding specificity of RPE65 with respect to both retinoids and other isoprenoids in an effort to define the specificity of binding, and to begin the process of designing specific antagonists for it. The nature of the specificity directed toward the three main structural elements (retinoid, linker, and acyl moieties) in the tRE molecule is reported. In the all-trans-retinyl ester series, binding affinity increased as a function of the hydrophobicity of the fatty acyl group. In the linker region, binding affinities were little affected by amide, ketone, and ether replacements for the carboxy ester moiety of the naturally occurring tRE ligand. Finally, modifications in the all-trans-retinoid moiety are also tolerated. For example, E,E-farnesyl palmitate binds with approximately the same affinity as does all-trans-retinyl palmitate. Other isoprenoid analogues also bind, as do truncated retinoids in the beta-ionone series. Therefore, mRPE65 is a moderately specific retinoid binding protein directed at long chain all-trans-retinyl esters.

PHYLOGENETIC RELATIONSHIPS OF THE MARSUPIAL GENUS HYLADELPHYS BASED ON NUCLEAR GENE SEQUENCES AND MORPHOLOGY

Phylogenetic analyses of sequence data from the nuclear gene encoding the interphotoreceptor retinoid–binding protein ( Irbp ) provide compelling evidence that the recently described Amazonian marsupial genus Hyladelphys is a didelphid and suggest that it occupies an internal branch separating the traditionally recognized subfamilies Caluromyinae and Didelphinae. Although this phylogenetic position also is supported by morphological character data, analyses of sequence data from the dentin matrix protein 1 gene ( Dmpl ) place Hyladelphys within the didelphine radiation. A parsimony analysis of the combined morphological and molecular data supports the Irbp -only results, but a partitioned Bayesian analysis of the combined gene data does not provide strong support for either placement. The implications of these results are discussed in terms of long-branch attraction, base-compositional bias, and other possibly confounding factors. Whether Hyladelphys is the sister group of Didelphinae or an independent lineage at some basal level within that subfamily, the absence of any close relationship to other Recent taxa is clearly indicated. We redescribe the genus based on new morphological character data and comment on the probable existence of undescribed taxa in addition to H. kalinowskii , the type species.

Antigen/MHC Class II/Ig Dimers for Study of Uveitogenic T Cells: IRBP p161–180 Presented by both IA and IE Molecules

Detection and modulation of effector T cells specific to immunodominant epitopes is a central issue in autoimmune diseases. Experimental autoimmune uveitis is a model for human autoimmune uveitis, induced in B10.RIII mice with interphotoreceptor retinoid binding protein or with its immunodominant epitope encoded by residues 161-180. The authors generated a dimer composed of p161-180 fused in frame to IA(r) and mouse IgG1, and studied its effects on a CD4(+) uveitogenic T-cell line specific to p161-180 and on a T-cell clone derived from that line. Immunofluorescent staining of the T-cell line with the peptide/IA(r)/Ig dimer revealed that about 90% of the cells bound the reagent, and 10% did not. The T-cell clone failed to bind the reagent. Consistent with this, the line proliferated when stimulated with the reagent plus anti-CD28, and the clone did not. Conversely, after being incubated with the reagent without CD28 cross-linking, the line showed decreased proliferation on subsequent stimulatory exposure to p161-180, whereas the clone was unaffected. Antigen-specific proliferation of splenocytes from B10.RIII mice primed with p161-180 was inhibited by anti-IA as well as anti-IE antibodies; proliferation of the T-cell line was inhibited strongly by anti-IA and poorly by anti-IE, and the clone showed the opposite pattern. Finally, the line, but not the clone, proliferated to p161-180 presented on a B-cell lymphoma expressing IA(r) as its only restriction element. Uveitogenic T cells can be detected as well as functionally modulated with their cognate peptide-class II reagent, suggesting the potential of such reagents for diagnostic and therapeutic use in uveitic disease; p161-180 can be presented by IA(r) as well as IE(r) major histocompatibility complex (MHC) class II molecules. The possibility that the same immunodominant fragment might be presented by more than one class II molecule should be taken into account when diagnostic or clinical use of peptide-MHC reagents is considered.

Ligand Recognition by RAR and RXR Receptors: Binding and Selectivity

Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

Genetic deficiency of C3 as well as CNS-targeted expression of the complement inhibitor sCrry ameliorates experimental autoimmune uveoretinitis

In this report, we describe the effect of complement deficiency and inhibition on experimental autoimmune uveoretinitis (EAU). C57BL/6 mice genetically deficient in C3 (C3−/−) or expressing a soluble complement activation inhibitor (soluble complement receptor related protein Y or sCrry) in a CNS-targeted fashion, (sCrry/GFAP) were induced for EAU via peripheral immunisation with a peptide of amino acids 1–20 of human interphotoreceptor retinoid binding protein in complete Freund's adjuvant with concurrent intraperitoneal pertussis toxin. The incidence and severity of EAU in the mutant mice was compared with that in simultaneously induced C57BL/6 wild type mice. The sCrry protein was detected in retinal extracts from transgenic but not wild type mice by western blot. C3−/− mice had a significant reduction in the incidence of EAU compared with wild type mice (incidence 44 versus 89%, respectively, p=0.0417) and a significant reduction in the severity of EAU (median disease score values 0 versus 1.3, respectively, p=0.0253). Similarly, sCrry mice had a significant reduction in the incidence of EAU compared with wild type mice (incidence 57 versus 100% respectively, p=0.0033) and a significant reduction in the severity of EAU (median disease score values 0.18 versus 1.85, respectively, p=0.0054). A genetic deficiency of C3 and production of a soluble complement inhibitor targeted to the CNS and eye are protective against EAU.

Structural insights into the cellular retinaldehyde‐binding protein (CRALBP)

Cellular retinaldehyde-binding protein (CRALBP) is an essential protein in the human visual cycle without a known three-dimensional structure. Previous studies associate retinal pathologies to specific mutations in the CRALBP protein. Here we use homology modeling and molecular dynamics methods to investigate the structural mechanisms by which CRALBP functions in the visual cycle. We have constructed two conformations of CRALBP representing two states in the process of ligand association and dissociation. Notably, our homology models map the pathology-associated mutations either directly in or adjacent to the putative ligand-binding cavity. Furthermore, six novel residues have been identified to be crucial for the hinge movement of the lipid-exchange loop in CRALBP. We conclude that the binding and release of retinoid involve large conformational changes in the lipid-exchange loop at the entrance of the ligand-binding cavity.

Retinoid status and responsiveness to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in mice lacking retinoid binding protein or retinoid receptor forms

We have investigated the role of Vitamin A (retinoid) proteins in hepatic retinoid processing under normal conditions and during chemical stress induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), a chemical known to interfere with retinoid turnover and metabolism. Three separate studies were performed in wildtype control mice and transgenic mice that lack one or more isoforms of retinoic acid receptors (RAR), retinoid X receptors (RXR), or intracellular retinoid-binding proteins (CRABP I, CRABP II, CRBP I). Body and organ weight development was monitored from 2 weeks of age to adult, and hepatic levels of retinyl esters, retinol, and retinoic acid were investigated. In addition, hepatic concentrations of 9- cis-4- oxo-13,14-dihydro-retinoic acid, a recently discovered retinoid metabolite that has proven sensitive to both TCDD exposure and Vitamin A status, were also determined. Mice absent in the three proteins CRBP I, CRABP I, and CRABP II (CI/CAI/CAII −/−) displayed significantly lower hepatic retinyl ester, retinol, and all- trans-retinoic acid levels compared to wildtype mice, whereas the liver concentrations of 9- cis-4- oxo-13,14-dihydro-retinoic acid was considerably higher. After treatment with TCDD, hepatic total retinoids were almost entirely depleted in the CI/CAI/CAII −/− mice, whereas wildtype mice and mice lacking CRABP I, and CRABP II (CAI/CAII −/−) retained approximately 60–70% of their Vitamin A content compared to controls at 28 days. RAR and RXR knockout mice responded similarly to wildtype mice with respect to TCDD-induced retinoid disruption, with the exception of RXRβ −/− mice which showed no decrease in hepatic Vitamin A concentration, suggesting that the role of RXRβ in TCDD-induced retinoid disruption should be further investigated. Overall, the abnormal retinoid profile in the triple knockout mice (CI/CAI/CAII −/−), but not double knockout (CAI/CAII −/−) mice, suggests that a loss of CRBP I may account for the difference in retinoid profile in CI/CAI/CAII −/− mice, and is likely to result in an increased susceptibility to hepatic retinoid depletion following dioxin exposure.

Disruption of CD40/CD40-Ligand Interactions in a Retinal Autoimmunity Model Results in Protection without Tolerance

We examined the role of CD40/CD40L interactions on the development of experimental autoimmune uveoretinitis (EAU), a cell-mediated, Th1-driven autoimmune disease that serves as a model for autoimmune uveitis in humans. EAU-susceptible B10.RIII mice immunized with the retinal autoantigen interphotoreceptor retinoid binding protein in CFA and treated with anti-CD40L Ab (MR1) had reduced incidence and severity of disease. Real-time PCR analysis revealed that the innate and adaptive responses of protected mice were reduced, without an obvious shift toward a Th2 cytokine profile. In contrast to some other reports, no evidence was found for regulatory cells in adoptive transfer experiments. To determine whether CD40L blockade resulted in long-term tolerance, mice protected by treatment with MR1 Ab were rechallenged for uveitis after circulating MR1 Ab levels dropped below the detection limit of ELISA. MR1-treated mice developed severe EAU and strong cellular responses to interphotoreceptor retinoid binding protein, comparable to those of control mice. These responses were higher than in mice that had not received the primary immunization concurrently with anti-CD40L treatment. We conclude that 1) CD40/CD40L interaction is required for EAU and its disruption prevents disease development; 2) CD40L blockade inhibits the innate response to immunization and reduces priming, but does not result in immune deviation; and 3) protection is dependent on persistence of anti-CD40L Abs, and long-term tolerance is not induced. Furthermore, immunological memory develops under cover of CD40L blockade causing enhanced responses upon rechallenge. Taken together, our data suggest that ongoing CD40/CD40L blockade might be required to maintain a therapeutic effect against uveitis.

Autoantigens signal through chemokine receptors: uveitis antigens induce CXCR3- and CXCR5-expressing lymphocytes and immature dendritic cells to migrate

AbstractWe tested the hypothesis that interaction between autoantigens and chemoattractant receptors may be an important step in the development of autoimmunity. The retinal autoantigens S-antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP) can induce autoimmune uveitis in rodent models. We evaluated the chemotactic activity of S-Ag and IRBP and found that both induced migration of human and mouse immature dendritic cells (iDCs) and lymphocytes, but not neutrophils, monocytes, or mature DCs. Cross-desensitization studies and single-receptor transfected cells revealed that subfamily of alpha chemokine receptors CXCR5 and CXCR3 mediated the chemotactic effect of IRBP, while only CXCR3 was required for the chemotactic response to S-Ag. Examination of the relationships between chemoattraction and the ability to elicit pathology at the protein or peptide levels in the mouse uveitis model revealed dissociation of the capacity to induce uveitis, lymphocyte proliferation, and chemoattraction. These studies suggest that IRBP and S-Ag can initiate innate and, in sensitive individuals, adaptive immune response by attracting iDCs and T and B cells expressing CXCR3 and CXCR5.

Identification and characterization of the retinoic acid response elements in the human RIG1 gene promoter

The expression of retinoic acid-induced gene 1 ( RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all- trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5′-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the −4910/−5509 fragment of the 5′-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between −5048 and −5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5′-TGACCTctattTGCCCT-3′ (DR5, −5243/−5259), and an inverted repeat sequence with six nucleotide spacing, 5′-AGGCCAtggtaaTGGCCT-3′ (IR6, −5323/−5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the atRA-mediated induction. Taken together, we have identified and characterized the functional atRA response element that is responsible for the atRA-mediated induction of RIG1 gene.

New DNA data from a transthyretin nuclear intron suggest an Oligocene to Miocene diversification of living South America opossums (Marsupialia: Didelphidae)

Phylogenetic relationships of 19 species of didelphid marsupials were studied using two nuclear markers, the non-coding transthyretin intron 1 (TTR) and the coding interphotoreceptor retinoid binding protein exon 1 (IRBP), and two mitochondrial genes, the protein-coding cytochrome b (cyt- b) and the structural 12S ribosomal DNA (12S rDNA). Evolutionary dynamics of these four markers were compared to each other, revealing the appropriate properties presented by TTR intron 1 together with its well supported and resolved phylogenetic signal. Nuclear markers supported the monophyly of medium and large-sized opossums Metachirus + ( Chironectes, Lutreolina, Didelphis, Philander), and the paraphyly of mouse-sized opossums, with the genera Gracilinanus, Thylamys, and Marmosops as a sister group to medium and large-sized didelphids. Conflicting branching patterns between mitochondrial and nuclear data involved the phylogenetic position of Marmosa– Micoureus– Monodelphis relative to other mouse-sized opossums. Nuclear phylogenetic inferences among genera were confirmed by the presence of synapomorphic indels observed in TTR intron 1. A Bayesian relaxed molecular clock dating of didelphid evolution using nuclear markers estimated their origin in the Middle Eocene (39.8 million years ago), with subsequent diversification during the Oligocene (Deseadan) and Miocene.

Temporal Changes in Gene Expression in the Arcuate Nucleus Precede Seasonal Responses in Adiposity and Reproduction

In anticipation of seasonal climate changes, Siberian hamsters display a strategy for survival that entails profound physiological adaptations driven by photoperiod. These include weight loss, reproductive quiescence, and pelage growth with shortening photoperiod and vice versa with lengthening photoperiod. This study reports gene expression changes in the hypothalamus of Siberian hamsters switched from short days (SD) to long days (LD), and also in photorefractory hamsters. Siberian hamsters were maintained in either LD or SD for 14 wk, conditions that generate physiological states of obesity under LD and leanness under SD. After 14 wk, SD lighting was switched to LD and gene expression investigated after 0, 2, 4, and 6 wk by in situ hybridization. Genes encoding nuclear receptors (RXR/RAR), retinoid binding proteins (CRBP1 and CRABP2), and histamine H3 receptor were photoperiodically regulated with significantly lower expression in SD, whereas VGF mRNA expression was significantly higher in SD, in the dorsomedial posterior arcuate nucleus. After a SD-to-LD switch, gene expression changes of CRABP2, RAR, H3R, and VGF occurred relatively rapidly toward LD control levels, ahead of body weight recovery and testicular recrudescence, whereas CRBP1 responded less robustly and rxrgamma did not respond at the mRNA level. In this brain nucleus in photorefractory animals, the CRABP2, RAR, H3R, and VGF mRNA returned toward LD levels, whereas CRBP1 and rxrgamma remained at the reduced SD level. Thus, genes described here are related to photoperiodic programming of the neuroendocrine hypothalamus through expression responses within a subdivision of the arcuate nucleus.

Experimental Autoimmune Uveitis Induced by Immunization with Retinal Pigment Epithelium–Specific 65-kDa Protein Peptides

Purpose: To investigate the pathogenic potential and sites of retinal pigment epithelium–specific 65-kDa protein (RPE65) for inducing experimental autoimmune uveitis (EAU) in Lewis rats. Methods: Twenty-six peptides were chemically synthesized based on the amino acid sequences of human RPE65. These peptides spanned the entire RPE65 sequence. Each peptide was injected into a footpad and the peritoneum of Lewis rats. The eyes were examined by slit-lamp biomicroscopy, and the findings were correlated with the histological findings. The serum antibody titer and lymphocyte reactivity against each peptide was also determined by enzyme-liked immunosorbent assay (ELISA) and lymphocyte proliferation assay, respectively. Results: Active immunization of rats resulted in the induction of EAU with 14 (3 severe and 11 mild) of the 26 peptides. The clinical course of the EAU was similar to that induced by the injection of retinal antigens such as S-antigen or inter-photoreceptor retinoid binding protein (IRBP). However, the histopathologic changes differed from the EAU induced by these retinal antigens. The inflammation was induced mainly from the retinal pigment epithelium (RPE) and the choroid, while the retina was relatively well-preserved except for some granulomatous changes adjacent to the RPE. Conclusions: Active immunization with peptides making up RPE65 will induce EAU. RPE65 has multiple EAU-inducing sites for Lewis rats.

Preliminary genetic characterization of two lineages of black rats (Rattus rattus sensu lato) in Japan, with evidence for introgression at several localities

We conducted a pilot survey of genetic diversity among 37 karyotyped individuals of the black rat Rattus rattus (sensu lato) from six localities on the Japanese Islands, using complete gene sequences of mitochondrial cytochrome b (cyt b) and nuclear interphotoreceptor retinoid binding protein (IRBP). Our sampling included two previously documented karyotypic groups: 'Oceanian' with 2n = 38 and 'Asian' with 2n = 42. Cyt b sequences for most individuals clustered according to their karyotypic groups, with an average between-group divergence of 3.8%. One exception was that individuals from Kagoshima (Kyushu Island) showed 'Asian' karyotypes combined with a cyt b haplotype that differed by a single nucleotide substitution from the haplotype of the 'Oceanian' karyotypic group. Six IRBP haplotypes were identified. They belonged to three distinct IRBP lineages (I-III), with an average inter-lineage divergence of 1%. Among homozygous individuals, these lineages showed good association with the karyotypic groups: IRBP lineage I occurred only with 'Oceanian' karyotypes, while IRBP lineages II and III both occurred with 'Asian' karyotypes. Individuals from Kagoshima all possessed IRBP of 'Asian' lineages, despite the presence of an 'Oceanian' mitochondrial type. The Chichijima population (Ogasawara Islands) featured exclusively 'Asian' karyotypes and cyt b sequences, but various combinations of all three IRBP lineages. The Kagoshima and Chichijima populations thus provide strong evidence of viable hybridization and genetic introgression between the two karyotypic groups, but with variable genetic outcomes. Our results demonstrate the potential of combined analysis of karyotypes and mitochondrial and nuclear gene sequences to elucidate the complex dispersal and population history of the black rat.

Behcet’s disease

Behcet's disease is a recurrent multisystem vasculitis that can affect any organ or system, but was originally described as orogenital ulcerations and uveitis. Specific criteria have been proposed for diagnosis and identification of affected organs by different national authorities. Behcet's disease is proposed to be due to an antigen/antibody reaction. The antigen can be external (microbial or other) or self-antigen. Self-antigens include HLA-B, S and interphotoreceptor retinoid binding protein, the oral mucosal antigens, and alphatropomycin. The antibody reaction manifests as changes common to any inflammatory process. Circulating immune complexes and neutrophil hyperactivity are present with the expression of adhesion molecules related to disease activity associated with phenotypic and functional aberrations affecting both Th1 and Th2 activity. The results in an increase of interleukin-4, -10, and -1B and interleukin-2 receptors with deficient interleukin-12, interferon-gamma, and expression of cell adhesion CDII/CD18, accompanied by increased B cells secreting IgG. The clinical picture varies according to the organ affected; emphasis is placed on those of diagnostic importance and those affecting morbidity and mortality. The histopathology demonstrates vasculitis with inflammatory cell infiltration, the endothelium expressing ICAM-1 and E-selectin, vascular occlusion with sequences. Treatment includes corticosteroids. Other immunosuppressant and biological agents include anti-tumor necrosis factor, interferon-alpha, Campath-1, and the S antigen. Colchicine and other miscellaneous therapies are included.

Physiological and microfluorometric studies of reduction and clearance of retinal in bleached rod photoreceptors.

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after ∼30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.

A Palmitoylated Light Switch

Vision depends on the activation of rhodopsin by light: The covalently bound rhodopsin antagonist 11- cis -retinal absorbs light, which triggers its irreversible photoisomerization to the all- trans agonist form and initiates a conformational change that allows rhodopsin to stimulate transducin. All- trans -retinal dissociates from opsin and is reduced to all- trans -retinol (vitamin A); lecithin retinol acyl transferase (LRAT) esterifies vitamin A to create all- trans -retinyl esters (mostly palmitate), which bind to a high-molecular-weight membrane-associated form of the retinal pigment epithelium protein RPE65 (mRPE65). mRPE65 mobilizes all- trans -retinyl esters, enabling their conversion into 11- cis -retinol and thereby allowing 11- cis -retinal to be regenerated and vision to continue. 11- cis -Retinol is regenerated more rapidly in the light; however, the mechanisms by which light regulates this visual cycle are poorly understood. Xue et al. used a fluorometric assay to show that soluble RPE65 (sRPE65) displayed complementary retinoid binding specificity to mRPE65, binding vitamin A with much higher affinity than all- trans -retinyl-palmitate and facilitating its conversion to all- trans -retinyl-palmitate. mRPE65 expressed in insect cells was labeled when exposed to 3 H-palmitate, and mass spectroscopic analysis indicated that mRPE65 was triply palmitoylated whereas sRPE65 was not palmitoylated. LRAT reversibly transferred a palmitoyl moiety from mRPE65 to vitamin A, generating sRPE65 and all- trans -retinyl palmitate. Moreover, 11- cis -retinol served as a substrate for mRPE65- and LRAT-mediated palmitoylation as well, so that 11- cis -retinol added to retinal pigment epithelium membranes promoted mRPE65 conversion to sRPE65 and thereby inhibited its own biosynthesis. Thus, the authors propose that palmitoylation of RPE65 provides a molecular switch regulating 11- cis -retinal synthesis, which can be flipped by the light-dependent increase in the supply of all- trans -retinyl esters and the ensuing conversion of sRPE65 to mRPE65. L. Xue, D. R. Gollapalli, P. Maiti, W. J. Jahng, R. R. Rando, A palmitoylation switch mechanism in the regulation of the visual cycle. Cell 117 , 761-771 (2004). [Online Journal]

Identification of CRALBP Ligand Interactions by Photoaffinity Labeling, Hydrogen/Deuterium Exchange, and Structural Modeling

Cellular retinaldehyde-binding protein (CRALBP) functions in the retinal pigment epithelium (RPE) as an acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a substrate carrier for 11-cis-retinol dehydrogenase. Toward a better understanding of CRALBP function, the ligand binding cavity in human recombinant CRALBP (rCRALBP) was characterized by photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal and by high resolution mass spectrometric topological analyses. Eight photoaffinity-modified residues were identified in rCRALBP by liquid chromatography tandem mass spectrometry, including Tyr(179), Phe(197), Cys(198), Met(208), Lys(221), Met(222), Val(223), and Met(225). Multiple different adduct masses were found on the photolabeled residues, and the molecular identity of each modification remains unknown. Supporting the specificity of photo-labeling, 50% of the modified residues have been associate with retinoid interactions by independent analyses. In addition, topological analysis of apo- and holo-rCRALBP by hydrogen/deuterium exchange and mass spectrometry demonstrated residues 198-255 incorporate significantly less deuterium when the retinoid binding pocket is occupied with 11-cis-retinal. This hydrophobic region encompasses all but one of the photo-labeled residues. A structural model of CRALBP ligand binding domain was constructed based on the crystal structures of three homologues in the CRAL-TRIO family of lipid-binding proteins. In the model, all of the photolabeled residues line the ligand binding cavity except Met(208), which appears to reside in a flexible loop at the entrance/exit of the ligand cavity. Overall, the results expand to 12 the number of residues proposed to interact with ligand and provide further insight into CRALBP ligand and protein interactions.