Cardioprotective efficacy of Xin-shu-bao tablet in heart failure with reduced ejection fraction by modulating THBD/ARRB1/FGF1/STIM1 signaling.

Traditional Chinese medicine offer unique advantages in mitigating and preventing early or intermediate stage for treating heart failure (HF). The purpose of this study was to assess the in vivo therapeutic efficacy of Xin-shu-bao (XSB) at different stages of HF following induction of a myocardial infarction (MI) in mice and use mass spectrometry-based proteomics to identify potential therapeutic targets for different stages of HF based on the molecular changes following XSB treatment. XSB had high cardioprotective efficacy in the pre-HF with reduced ejection fraction (HFrEF) stages, but had a weak or no effect in the post-HFrEF stages. This was supported by echocardiographic measurements showing that XSB decreased ejection fraction and fractional shortening in HF. XSB administration improved cardiac function in the pre- and post-HFrEF mouse model, ameliorated deleterious changes to the morphology and subcellular structure of cardiomyocytes, and reduced cardiac fibrosis. Proteomics analysis showed that XSB intervention exclusively targeted thrombomodulin (THBD) and stromal interaction molecule 1 (STIM1) proteins when administered to the mice for both 8 and 6 weeks. Furthermore, XSB intervention for 8, 6, and 4 weeks after MI induction increased the expression of fibroblast growth factor 1 (FGF1) and decreased arrestin ß1 (ARRB1), which are classic biomarkers of cardiac fibroblast transformation and collagen synthesis, respectively. Overall, the study suggests that early intervention with XSB could be an effective strategy for preventing HFrEF and highlights potential therapeutic targets for further investigation into HFrEF remediation strategies.

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis.

BACKGROUND: Increasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. PURPOSE: The purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. METHODS: A gaussia princeps luciferase-based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and ß-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. RESULTS: Celastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. CONCLUSION: Taken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.

FFA4 receptor (GPR120): A hot target for the development of anti-diabetic therapies.

Free Fatty Acid 4 receptor (FFA4 receptor or GPR120), a rhodopsin-like G protein coupled receptor (GPCR) subfamily member, is a receptor that senses specific fatty acids such as ω-3 fatty acid in fish oil or the endogenous signaling lipid, PHASA. FFA4 receptor is enriched in lung, colon and adipose tissue but is also detected in many other tissues and cells. The activation of FFA4 receptor has multiple effects, including but not limited to inhibition of inflammation, improving insulin sensitivity and adipogenesis, and regulating hormone secretion from the gastro-intestinal system and pancreatic islets. The important role of FFA4 receptor in maintaining metabolic homeostasis strongly indicates the great potential of selective FFA4 receptor agonizts to treat diabetes and inflammation. In this review, we summarize recent research progress in the physiological and biochemical studies of FFA4 receptor and highlight its underlying signaling mechanisms and ligand identification to assist future research to exploit FFA4 receptor as a drug target.

Molecular determinants of orexin receptor-arrestin-ubiquitin complex formation.

BACKGROUND AND PURPOSE: The orexin system regulates a multitude of key physiological processes, particularly involving maintenance of metabolic homeostasis. Consequently, there is considerable potential for pharmaceutical development for the treatment of disorders from narcolepsy to metabolic syndrome. It acts through the hormonal activity of two endogenous peptides, orexin A binding to orexin receptors 1 and 2 (OX1 and OX2) with similar affinity, and orexin B binding to OX2 with higher affinity than OX1 receptors. We have previously revealed data differentiating orexin receptor subtypes with respect to their relative stability in forming orexin receptor-arrestin-ubiquitin complexes measured by BRET. Recycling and cellular signalling distinctions were also observed. Here, we have investigated, using BRET, the molecular determinants involved in providing OX2 receptors with greater ß-arrestin-ubiquitin complex stability. EXPERIMENTAL APPROACH: The contribution of the C-terminal tail of the OX receptors was investigated by bulk substitution and site-specific mutagenesis using BRET and inositol phosphate assays. KEY RESULTS: Replacement of the OX1 receptor C-terminus with that of the OX2 receptor did not result in the expected gain of function, indicating a role for intracellular domain configuration in addition to primary structure. Furthermore, two out of the three putative serine/threonine clusters in the C-terminus were found to be involved in OX2 receptor-ß-arrestin-ubiquitin complex formation. CONCLUSIONS AND IMPLICATIONS: This study provides fundamental insights into the molecular elements that influence receptor-arrestin-ubiquitin complex formation. Understanding how and why the orexin receptors can be functionally differentiated brings us closer to exploiting these receptors as drug targets.

Novel middle-type Kenyon cells in the honeybee brain revealed by area-preferential gene expression analysis.

The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel 'middle-type Kenyon cells' that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large- and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large- and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large- and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a 'clustering' of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.

Arrestin-dependent but G-protein coupled receptor kinase-independent uncoupling of D2-dopamine receptors.

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (K(ir)3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-K(ir)3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.

Vitamin A supplementation ameliorates obesity-associated retinal degeneration in WNIN/Ob rats.

OBJECTIVE: Obesity is associated with various health afflictions, including ocular complications such as diabetic retinopathy, high intraocular pressure, cataracts, and macular degeneration. We previously reported progressive retinal degeneration after the onset of obesity in the spontaneously obese rat (WNIN/Ob) model. In the present study, we investigated vitamin A supplementation to ameliorate obesity-associated retinal degeneration in the WNIN/Ob rat. METHODS: Five-month-old male WNIN/Ob obese (O) and lean (L) control rats were fed with vitamin A 2.6 mg (L/O-I), 26 mg (L/O-II), 52 mg (L/O-III), and 129 mg (L/O-IV) per kilogram of diet as retinyl palmitate for 4 mo 2 wk. Retinal morphology and retinal gene expression were assessed by histologic, immunohistochemical, and real-time polymerase chain reaction methods. RESULTS: Supplementation of vitamin A at 26 or 52 mg significantly modulated the expression of retinal genes in the O but not in the L phenotype. Vitamin A supplementation significantly upregulated the expression of genes, such as rhodopsin, rod arrestin, phosphodiesterase, transducins, and fatty acid elongase-4, that were otherwise downregulated in O rat retina. The expression of glial fibrillary acidic protein was downregulated by vitamin A feeding in O rat retina. The immunohistochemical and histologic findings corroborated the gene expression data. The effects were significant at a 26- or 52-mg dose of vitamin A. CONCLUSION: Vitamin A supplementation alleviated obesity-associated retinal degeneration in the WNIN/Ob rat.

Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo.

The present study describes a robust 50-fold increase in rhodopsin gene transcription by cAMP in cultured retinal precursor cells of chicken embryo. Retinal cells isolated at embryonic day 8 (E8) and cultured for 3 days in serum-supplemented medium differentiated mostly into red-sensitive cones and to a lesser degree into green-sensitive cones, as indicated by real-time RT-PCR quantification of each specific opsin mRNA. In contrast, both rhodopsin mRNA concentration and rhodopsin gene promoter activity required the presence of cAMP-increasing agents [forskolin and 3-isobutyl-1-methylxanthine (IBMX)] to reach significant levels. This response was rod-specific and was sufficient to activate rhodopsin gene transcription in serum-free medium. The increase in rhodopsin mRNA levels evoked by a series of cAMP analogs suggested the response was mediated by protein kinase A, not by EPAC. Membrane depolarization by high KCl concentration also increased rhodopsin mRNA levels and this response was strongly potentiated by IBMX. The rhodopsin gene response to cAMP-increasing agents was developmentally gated between E6 and E7. Rod-specific transducin alpha subunit mRNA levels also increased up to 50-fold in response to forskolin and IBMX, while rod-specific phosphodiesterase-VI and rod arrestin transcripts increased 3- to 10-fold. These results suggest a cAMP-mediated signaling pathway may play a role in rod differentiation.

Mitochondrial oxidative DNA damage in experimental autoimmune uveitis.

PURPOSE: In experimental autoimmune uveitis (EAU), recent work has demonstrated that retinal damage involves oxidative stress early in uveitis, before macrophage cellular infiltration. The purpose of this study was to determine whether oxidative mitochondrial DNA damage occurs early in EAU, before leukocyte infiltration. METHODS: Lewis rats were immunized with S-antigen mixed with complete Freund adjuvant (CFA) to induce EAU. Nonimmunized animals and animals injected with CFA served as controls. Animals were killed on days 3, 4, 7, and 12 after immunization. Damage to mitochondrial DNA and nuclear DNA was assessed using a novel long quantitative polymerase chain reaction technique. TUNEL staining to detect apoptosis and immunohistochemical detection of leukocyte infiltration in EAU retinas were also performed at these times. RESULTS: Mitochondrial DNA damage occurred early in EAU, from day 4 to day 12. In the early phase of EAU (days 4-7), there was no inflammatory cell infiltration. On day 12 inflammatory cells infiltrated the retina and uvea. Nuclear DNA damage occurred later in EAU at day 12. Neither mitochondrial nor nuclear DNA damage was detected in the controls. TUNEL-positive staining for apoptosis was detected only at day 12 in EAU retina. CONCLUSIONS: Oxidative mitochondrial DNA damage begins at day 4 in EAU, supporting the view that oxidative stress selectively occurs in the mitochondria in the early phase of EAU, before leukocyte infiltration. Such oxidative damage in the mitochondria may be the initial event leading to retinal degeneration in EAU.

Identification of MHC class I H-2 Kb/Db-restricted immunogenic peptides derived from retinal proteins.

PURPOSE: To identify H-2 Kb/Db-binding immunogenic peptides derived from retinal proteins. METHODS: Computer-based prediction was used to identify potentially H-2 Kb/Db-binding peptides derived from the interphotoreceptor retinol-binding protein (IRBP), soluble retinal antigen (S-antigen), recoverin, phosducin, and pigment epithelium-derived factor (PEDF). The affinity of the peptides was analyzed by their abilities to upregulate the expression of major histocompatibility complex (MHC) class I on TAP-deficient cells (RMA-S cells) with flow cytometry. C57BL/6 mice were immunized subcutaneously, with individual peptides in incomplete Freund's adjuvant (IFA). Eight days after immunization, splenocytes were isolated for cytotoxic T-lymphocyte (CTL) analysis. A 51chromium-release assay was used to detect specific CTL reactivity generated in the cultures. Eyes were enucleated for histopathological analysis on day 21 after immunization with IRBP or IRBP and the immunogenic peptides. RESULTS: All the 21 predicted peptides were found to upregulate expression of H-2 Kb/Db on RMA-S cells. Five peptides, the two IRBP-derived peptides IRBP89-96 and IRBP(101-108), and the three PEDF-derived peptides, PEDF389-397, PEDF139-147, and PEDF272-279, induced specific CTL responses in vivo, whereas the remaining 16 peptides, including 5 IRBP-derived peptides, 5 S-antigen-derived peptides, 1 recoverin-derived peptide, 1 phosducin-derived peptide, and 4 PEDF-derived peptides, did not induce specific CTL reactivity. The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. CONCLUSIONS: Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.

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.

N-formyl peptide receptors cluster in an active raft-associated state prior to phosphorylation.

In response to ligand binding, G protein-coupled receptors undergo phosphorylation and activate cellular internalization machinery. An important component of this process is the concentration of receptors into clusters on the plasma membrane. Aside from organizing the receptor in anticipation of internalization, little is known of the function of ligand-mediated G protein-coupled receptor clustering, which has traditionally been thought of as being a phosphorylation-dependent event prior to receptor internalization. We now report that following receptor activation, the N-formyl peptide receptor (FPR) forms distinct membrane clusters prior to its association with arrestin. To determine whether this clustering is dependent upon receptor phosphorylation, we used a mutant form of the FPR, DeltaST-FPR, which lacks all phosphorylation sites in the carboxyl-terminal domain. We found that activation of the signaling-competent DeltaST-FPR resulted in rapid receptor clustering on the plasma membrane independent of Gi protein activation. This clustering required receptor activation since the D71A mutant receptor, which binds ligand but is incapable of transitioning to an active state, failed to induce receptor clustering. Furthermore we demonstrated that FPR-mediated clustering and signaling were cholesterol-dependent processes, suggesting that translocation of the active receptor to lipid rafts may be required for maximal signaling activity. Finally we showed that FPR stimulation in the absence of receptor phosphorylation resulted in translocation of FPR to GM1-rich clusters. Our results demonstrate for the first time that formation of a clustered activated receptor state precedes receptor phosphorylation, arrestin binding, and internalization.

The uveitogenic potential of retinal S-antigen in horses.

PURPOSE: To investigate the uveitogenic potential of retinal S-antigen (S-Ag) in horses. METHODS: Horses were immunized subcutaneously with S-Ag or BSA as control antigen, emulsified in complete Freund's adjuvant. Simultaneously, Bordetella pertussis was given intravenously. Antigen specific T- and B-cell responses were analyzed in a 3-day interval. Disease development was judged clinically and histopathologically. Two identical booster immunizations were given every 4 weeks to test induction of recurrences. RESULTS: T- and B-cell responses specific for S-Ag were observed in all immunized horses but were absent in control animals. However, uveitis developed in only one of five animals. Reimmunization with S-Ag did not lead to a uveitic relapse in this horse. All other horses of the S-Ag- and BSA-treated groups neither showed any signs of uveitis, nor had inflammatory infiltrates of the inner eye. CONCLUSIONS: In contrast to interphotoreceptor retinoid-binding protein (IRBP), S-Ag is a weak autoantigen in horses. Even though S-Ag immunization leads to the activation of autoreactive T- and B-cells, infiltration of the inner eye and induction of uveitis are controlled in most horses.

Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability.

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.

Induction or suppression of a B cell-specific response to self antigen in vivo is dependent upon dendritic cell activation via the TNF-alpha receptor at the time of antigen uptake.

In this study we show that the retinal autoantigen, S-antigen, contains a functional TNF-alpha homologous domain which stimulates maturation and differentiation of cultured dendritic cells (DC) or tissue DC via the p55 TNF-alpha receptor. Tissue DC became more dendritiform in shape, and migrated into culture supernatant. S-antigen also stimulated accumulation of cell surface MHC class II antigen with a corresponding loss of acidic intracellular vesicles, and induced IL-1beta and IL-12 mRNA expression in cultured bone marrow-derived DC. In addition, cultured splenic DC primed immune responses to S-antigen in vivo in the absence of other, exogenous cytokine sources. DC pulsed with either retinal S-antigen or another retinal autoantigen, interphotoreceptor retinoid binding protein (IRBP), were able to stimulate naive T cell proliferation in vitro, but only S-antigen-pulsed DC were able to induce an immune response in vivo and initiate antibody class switching. In contrast, IRBP-pulsed DC had no detectable in vivo priming effect and IgG antibody levels remained suppressed even after immunization with IRBP in complete Freund's adjuvant. These results indicate that DC from the same precursor population can either induce or suppress a B cell-specific response to self antigen in vivo, the outcome being dependent upon DC activation at the time of antigen uptake and presentation.

A novel non-polyglutamable anti-folate, MX-68, inhibits the induction of experimental autoimmune uveitis in rats.

MX-68 is a novel antifolate which is chemically designed not to undergo intracellular polyglutamation thus preventing the development of adverse effects. The present study was carried out to examine both the in vitro and in vivo effects of MX-68 on experimental autoimmune uveitis (EAU) and to compare its effect on collagen-induced arthritis (CIA) in rats. EAU was induced by injecting Lewis rats with retinal S-antigen in complete Freund's adjuvant. Either MX-68 or methotrexate (MTX), which forms several polyglutamates intracellularly, was orally administered five days a week for three weeks beginning on the day of immunization. In vivo, both MX-68 and MTX significantly delayed the onset of EAU and inhibited the antibody response to S-antigen in a dose-dependent manner. High dose MX-68 (2.5 mg kg-1 day-1) completely abrogated the induction of EAU. No adverse effects were observed in either MX-68- or MTX-treated rats. However, the cessation of MX-68 administration after a period of three weeks resulted in the induction of EAU. In contrast, both MX-68 and MTX suppressed the severity of CIA without affecting the onset of the disease and inhibited anti-collagen antibody production in a dose-dependent fashion. Discontinuation of the drugs did not result in the recurrence of CIA. In vitro, both MX-68 and MTX significantly suppressed the proliferation of S-antigen- and Con A-stimulated lymph node cells obtained from immunized rats in a dose-dependent fashion. These data suggest that MX-68 may be useful for the treatment of autoimmune diseases including EAU and that the pathophysiology of EAU could be different from that of CIA.

Immunomodulatory effects of linomide in animals immunized with immunopathogenic retinal antigens: dissociation between different immune functions.

Linomide (LS-2616, quinoline-3-carboxamide) has been reported to exert a diverse range of effects on the immune system. On one hand, this drug was found to stimulate the immune system and to enhance activities such as DTH or allograft rejection. On the other hand, linomide was shown to inhibit the induction of experimental autoimmune encephalomyelitis and myasthenia gravis, as well as the development of diabetes in non-obese diabetic (NOD) mice. Here we report the effects of linomide in animals immunized with uveitogenic retinal antigens. Treatment with linomide completely inhibited the development of experimental autoimmune uveoretinitis (EAU) in mice immunized with interphotoreceptor retinoid-binding protein and markedly suppressed EAU in rats immunized with S-antigen (S-Ag). In addition, linomide-treated rats exhibited reduced antibody production and lymphocyte proliferative response to S-Ag. In contrast to these suppressive activities, linomide treatment did not affect the development of adoptively transferred EAU in rats and moderately enhanced the DTH reactions to S-Ag in immunized rats in which EAU and other immune responses to this antigen were suppressed.

Calcium/calmodulin-dependent protein kinase II and arrestin phosphorylation in Limulus eyes.

In rhabdomeral photoreceptors, light stimulates the phosphorylation of arrestin, a protein critical for quenching the photoresponse, by activating a calcium/calmodulin-dependent protein kinase (CaM PK). Here we present biochemical evidence that a CaM PK that phosphorylates arrestin in Limulus eyes is structurally similar to mammalian CaM PK II. In addition, cDNAs encoding proteins homologous to mammalian and Drosophila CaM PK II in the catalytic and regulatory domains were cloned and sequenced from a Limulus lateral eye cDNA library. The Limulus sequences are unique, however, in that they lack most of the association domain. The proteins encoded by these sequences may phosphorylate arrestin.

Isolation and expression of an arrestin cDNA from the horseshoe crab lateral eye.

Electrophysiological studies of photoreceptors from the horseshoe crab Limulus polyphemus continue to provide fundamental new knowledge of the photoresponse in invertebrates. Therefore, it is of particular interest to characterize the molecular components of the photoresponse in this system. Here we describe an arrestin cloned from a cDNA library constructed using poly(A)+ RNA isolated from Limulus lateral eyes. The protein, deduced from the arrestin cDNA, is most similar to arrestin from locust antennae (56% identity) and Drosophila phosrestin I (53% identity). Limulus arrestin was expressed in a heterologous system, and its properties were compared with those of a 46-kDa light-regulated phosphoprotein (pp46A) in Limulus photoreceptors described in previous studies from this laboratory. Arrestin and pp46A (a) have the same apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) have an isoelectric point in the basic pH range, (c) require calmodulin and elevated Ca2+ levels for phosphorylation, (d) are immunoreactive with monoclonal antibody C10C10 directed against a sequence in bovine arrestin (S-antigen) that is perfectly conserved in the deduced arrestin protein, and (e) are associated with photoreceptors. We conclude that the arrestin described here and pp46A are the same protein. The results of this and previous studies show that in Limulus photoreceptors, light regulates the phosphorylation of arrestin in complex ways.