Method Development and Validation for Simultaneous Determination of Six Flavonoids in Rat Eyes after Oral Administration of Diospyros kaki Leaves Extract by UPLC-MS/MS.

A robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique was proven effective for simultaneous characterization of six flavonoids including quercetin-3-O-beta-galactoside (Q3GAL), quercetin-3-O-beta-glucoside (Q3GLU), quercetin-3-(2-galloylglucoside) (Q3GG), kaempferol-3-O-beta-galactoside (K3GAL), kaempferol-3-O-beta-glucoside (K3GLU), and kaempferol-3-(2-galloylglucoside) (K3GG) in rat eyes. By investigation of corresponding validation parameters (linearity, selectivity, precision, accuracy, matrix effect, extraction recovery, and stability), the method was verified to be within current acceptable criteria. Thereafter, the validated method enabled quantification of the six compounds successful in rat eyes after oral administration of ethanol extract Diospyros kaki (EEDK) at 0, 3, 15, 35, 60, 120 min.

A role for spindles in the onset of rapid eye movement sleep.

Sleep spindle generation classically relies on an interplay between the thalamic reticular nucleus (TRN), thalamo-cortical (TC) relay cells and cortico-thalamic (CT) feedback during non-rapid eye movement (NREM) sleep. Spindles are hypothesized to stabilize sleep, gate sensory processing and consolidate memory. However, the contribution of non-sensory thalamic nuclei in spindle generation and the role of spindles in sleep-state regulation remain unclear. Using multisite thalamic and cortical LFP/unit recordings in freely behaving mice, we show that spike-field coupling within centromedial and anterodorsal (AD) thalamic nuclei is as strong as for TRN during detected spindles. We found that spindle rate significantly increases before the onset of rapid eye movement (REM) sleep, but not wakefulness. The latter observation is consistent with our finding that enhancing spontaneous activity of TRN cells or TRN-AD projections using optogenetics increase spindle rate and transitions to REM sleep. Together, our results extend the classical TRN-TC-CT spindle pathway to include non-sensory thalamic nuclei and implicate spindles in the onset of REM sleep.

Prolonged intraocular residence and retinal tissue distribution of a fourth-generation compstatin-based C3 inhibitor in non-human primates.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss among the elderly population. Genetic studies in susceptible individuals have linked this ocular disease to deregulated complement activity that culminates in increased C3 turnover, retinal inflammation and photoreceptor loss. Therapeutic targeting of C3 has therefore emerged as a promising strategy for broadly intercepting the detrimental proinflammatory consequences of complement activation in the retinal tissue. In this regard, a PEGylated second-generation derivative of the compstatin family of C3-targeted inhibitors is currently in late-stage clinical development as a treatment option for geographic atrophy, an advanced form of AMD which lacks approved therapy. While efficacy has been strongly suggested in phase 2 clinical trials, crucial aspects still remain to be defined with regard to the ocular bioavailability, tissue distribution and residence, and dosing frequency of such inhibitors in AMD patients. Here we report the intraocular distribution and pharmacokinetic profile of the fourth-generation compstatin analog, Cp40-KKK in cynomolgus monkeys following a single intravitreal injection. Using a sensitive surface plasmon resonance (SPR)-based competition assay and ELISA, we have quantified both the amount of inhibitor and the concentration of C3 retained in the vitreous of Cp40-KKK-injected animals. Cp40-KKK displays prolonged intraocular residence, being detected at C3-saturating levels for over 3 months after a single intravitreal injection. Moreover, we have probed the distribution of Cp40-KKK within the ocular tissue by means of immunohistochemistry and highly specific anti-Cp40-KKK antibodies. Both C3 and Cp40-KKK were detected in the retinal tissue of inhibitor-injected animals, with prominent co-localization in the choroid one-month post intravitreal injection. These results attest to the high retinal tissue penetrance and target-driven distribution of Cp40-KKK. Given its subnanomolar binding affinity and prolonged ocular residence, Cp40-KKK constitutes a promising drug candidate for ocular pathologies underpinned by deregulated C3 activation.

Estimation of the Minimum Effective Dose of Dietary Supplement Crocetin for Prevention of Myopia Progression in Mice.

The natural carotenoid crocetin has been reported to suppress phenotypes of an experimental myopia model in mice. We investigated the minimum effective dose to prevent myopia progression in a murine model. Three-week-old male mice (C57B6/J) were equipped with a -30 diopter (D) lens to induce myopia, and fed with normal chow, 0.0003%, or 0.001% of crocetin-containing chow. Changes in refractive errors and axial lengths (AL) were evaluated after three weeks. Pharmacokinetics of crocetin in the plasma and the eyeballs of mice was evaluated with specific high sensitivity quantitative analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the minimum effective dosage. A concentration of 0.001% of crocetin-containing chow showed a significant (p < 0.001) suppressive effect against both refractive and AL changes in the murine model. Meanwhile, there was no significant difference of AL change between the 0.0003% and the normal chow groups. The concentration of crocetin in the plasma and the eyeballs from mice fed with 0.001% crocetin-containing chow was significantly higher than control and 0.0003% crocetin-containing chow. In conclusion, we suggest 0.001% of crocetin-containing extract is the minimum effective dose showing a significant suppressive effect against both refractive and AL changes in the murine model.

Skipjack tuna (Katsuwonus pelamis) eyeball oil exerts an anti-inflammatory effect by inhibiting NF-κB and MAPK activation in LPS-induced RAW 264.7 cells and croton oil-treated mice.

The effect of tuna eyeball oil (TEO) on lipopolysaccharide (LPS)-induced inflammation in macrophage cells was investigated. TEO had no cytotoxicity in cell viability as compared to the control in LPS induced RAW 264.7 cells. TEO reduced the levels of NO and pro-inflammatory cytokines by up to 50% in a dose-dependent manner. The expression of NF-κB and MAPKs as well as iNOS and COX-2 proteins was reduced by TEO, which suggests that its anti-inflammatory activity is related to the suppression of the NF-κB and MAPK signaling pathways. The rate of formation of ear edema was reduced compared to that in the control at the highest dose tested. In an acute toxicity test, no mice were killed by TEO doses of up to 5000mg/kg body weight during the two week observation period. These results suggested that TEO may have a significant effect on inflammatory factors and be a potential anti-inflammatory therapeutic.

Development of a method to quantify clindamycin in vitreous humor of rabbits' eyes by UPLC-MS/MS: application to a comparative pharmacokinetic study and in vivo ocular biocompatibility evaluation.

Ocular toxoplasmosis may result in uveitis in the posterior segment of the eye, leading to severe visual complications. Clindamycin-loaded poly(lactide-co-glycolide) (PLGA) implants could be applied to treat the ocular toxoplasmosis. In this study, the pharmacokinetic profiles of the drug administrated by PLGA implants and by intravitreal injections in rabbits' eyes were evaluated. The implant released the drug for 6 weeks while the drug administrated by intravitreal injections remained in the vitreous cavity for 2 weeks. Compared to the injected drug, the implants containing clindamycin had higher values of area under the curve (AUC) (39.2 vs 716.7 ng week mL(-1)) and maximum vitreous concentration (Cmax) (8.7 vs 13.83 ng mL(-1)). The implants prolonged the delivery of clindamycin and increased the contact of the drug with the eyes' tissues. Moreover, the in vivo ocular biocompatibility of the clindamycin-loaded PLGA implants was evaluated regarding to the clinical examination of the eyes and the measurement of the intraocular pressure (IOP) during 6 weeks. The implantable devices caused no ocular inflammatory process and induced the increase of the IOP in the fourth week of the study. The IOP augmentation could be related to the maximum concentration of clindamycin released from the implants. In conclusion, the PLGA implants based on clindamycin may be a therapeutic alternative to treat ocular toxoplasmosis.

Color variation assay of the anthocyanins from Açai Fruit (Euterpe oleracea): a potential new dye for vitreoretinal surgery.

AIM: The goals of this study were to determine the potential for use of the natural anthocyanins from the açai fruit (Euterpe oleracea) during vitreoretinal surgery and the ideal physicochemical properties of the dye. METHODS: We evaluated the color variations of the dye at different pHs and osmolarities with or without the use of mordants as a potential new tool for internal limiting membrane peeling. The extracts of anthocyanin from the açai fruit were analyzed by spectrophotometry to determine the degree of color variations associated with various pHs and osmolarities. The experiments were conducted in test tubes filled with tryptophan soya media and Petri dishes prepared with agar media. RESULTS: We observed various shades of green, red, and purple in the extracts of the anthocyanin dye at different pHs and osmolarities. The assay to adjust the anthocyanin solution similar to the physiologic retinal environment (osmolarity, 300 mOsm; pH, 7.00) resulted in a shade of purple that may be useful to stain the intraocular microstructures during vitreoretinal surgery. The physicochemical property of the purple anthocyanin solutions from the açai fruit was observed at physiologic pH and osmolarity. CONCLUSION: Anthocyanins from the açai fruit may be useful to enhance visualization of the intraocular microstructures during vitreoretinal surgery.

Positive moderation of the hematology, plasma biochemistry and ocular indices of oxidative stress in alloxan-induced diabetic rats, by an aqueous extract of the leaves of Sansevieria liberica Gerome and Labroy.

OBJECTIVE: To investigate the ability of an aqueous extract of the leaves of Sansevieria liberica (S. liberica) to alter the hematology, plasma biochemistry and ocular indices of oxidative stress in alloxan-induced diabetic rats. METHOD: Diabetes mellitus was induced by injection of alloxan (80 mg/kg body weight), via the tail vein. The extract was administered orally at 100, 200 and 300 mg/kg body weight (both to normal and diabetic rats), and metformin at 50 mg/kg body weight. RESULTS: Compared to test control, the treatment dose dependently, significantly lowered (P<0.05) ocular malondialdehyde content, atherogenic indices, red cell, total white cell and lymphocyte counts, mean cell hemoglobin concentration; and plasma levels of glucose, triglyceride, total-, very low density lipoprotein-, low density lipoprotein- and non-high density lipoprotein cholesterols, total, conjugated and unconjugated bilirubin, sodium, urea, blood urea nitrogen, as well as plasma activities of alkaline phosphatase, alanine and aspartate transaminases. However, the treatment significantly increased (P<0.05) hematocrit, hemoglobin concentration, mean cell hemoglobin, and mean cell volume, neutrophil and monocyte counts, and plasma levels of high density lipoprotein cholesterol, potassium, chloride, calcium, bicarbonate and total protein, ocular ascorbic acid content and ocular activities of catalase and superoxide dismutase. This study showed the hypoglycemic, hypolipidemic, immune-modulating, ocular-, hepato-renal and cardio-protective potentials of the extract. CONCLUSIONS: All these, support the use of the leaves of S. liberica in African traditional health care practices for the management of diabetes mellitus.

Biosynthesis of very long-chain fatty acids (C>24) in Atlantic salmon: cloning, functional characterisation, and tissue distribution of an Elovl4 elongase.

The elongases of very long-chain fatty acids (Elovl) account for the rate-limiting condensation step of the elongation process in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovl family, Elovl4, has been regarded as a critical enzyme in vertebrates in the production of the so-called very long-chain fatty acids (VLC-FA), a group of compounds that has been scarcely explored in fish. Here we report on the cloning of a novel Elovl4-like elongase from Atlantic salmon (Salmo salar). The salmon Elovl4 cDNA codes for a putative protein containing 306 amino acids. Heterologous expression in yeast demonstrated that salmon Elovl4 efficiently elongated saturated FAs up to 36:0, with 24:0 and 26:0 appearing as preferred substrates. Additionally, salmon Elovl4 effectively converted C20 and C22 polyunsaturated fatty acids to elongated polyenoic products up to C36. Tissue distribution showed that Elovl4 mRNA transcripts are abundant in eye, brain and testes, suggesting that, as described in mammals, these tissues are important metabolic sites for the biosynthesis of VLC-FA. Our results are discussed in comparison with the functional analyses observed in Elovl4 proteins from other vertebrates, and also other Elovl proteins investigated previously in Atlantic salmon.

Xanthophylls and eye health of infants and adults.

Lutein and zeaxanthin are the only carotenoids present in the eye. They cannot be synthesized de novo and are specifically concentrated in the macula. They appear to have at least two major functions: to filter out blue light and thus prevent ensuing damages to the eye and to act as antioxidants. Infants are particularly at risk from both blue light and oxidative damage to eye tissues. Lutein is present in human milk but is not currently added to infant formulas. Fortifying formulae with lutein in order to match more closely human milk might help protect the infant's sensitive eyes. In adults, the exact pathogenesis of age-related maculopathy remains unknown. Light damage, inflammation, and the disruption of cellular processes by oxidative stress may play an important role in the degenerative process. Manipulation of intake of xanthophylls has been shown to augment macular pigment, therefore it is thought that carotenoid dietary supplements could prevent, delay, or modify the course of age-related maculopathy. However, definite evidence of the effect of carotenoids, the optimal doses to use, and the supplementation duration are still under investigation.

Identification and metabolic transformations of carotenoids in ocular tissues of the Japanese quail Coturnix japonica.

As in humans and monkeys, lutein [(3R,3'R,6'R)-beta,epsilon-carotene-3,3'-diol] and zeaxanthin [a mixture of (3R,3'R)-beta,beta-carotene-3,3'diol and (3R,3'S-meso)-beta,beta-carotene-3,3'-diol] are found in substantial amounts in the retina of the Japanese quail Coturnix japonica. This makes the quail retina an excellent nonprimate small animal model for studying the metabolic transformations of these important macular carotenoids that are thought to play an integral role in protection against light-induced oxidative damage such as that found in age-related macular degeneration (AMD). In this study, we first identified the array of carotenoids present in the quail retina using C30 HPLC coupled with in-line mass spectral and photodiode array detectors. In addition to dietary lutein (2.1%) and zeaxanthin (11.8%), we identified adonirubin (5.4%), 3'-oxolutein (3.8%), meso-zeaxanthin (3.0%), astaxanthin (28.2%), galloxanthin (12.2%), epsilon,epsilon-carotene (18.5%), and beta-apo-2'-carotenol (9.5%) as major ocular carotenoids. We next used deuterium-labeled lutein and zeaxanthin as dietary supplements to study the pharmacokinetics and metabolic transformations of these two ocular pigments in serum and ocular tissues. We then detected and quantitated labeled carotenoids in ocular tissue using both HPLC-coupled mass spectrometry and noninvasive resonance Raman spectroscopy. Results indicated that dietary zeaxanthin is the precursor of 3'-oxolutein, beta-apo-2'-carotenol, adonirubin, astaxanthin, galloxanthin, and epsilon,epsilon-carotene, whereas dietary lutein is the precursor for meso-zeaxanthin. Studies also revealed that the pharmacokinetic patterns of uptake, carotenoid absorption, and transport from serum into ocular tissues were similar to results observed in most human clinical studies.

Preliminary study on Japanese cedar pollinosis in an artificial exposure chamber (OHIO Chamber).

BACKGROUND: A pollen exposure chamber (OHIO Chamber) was built in central Tokyo, Japan, in order to study seasonal allergic rhinitis (SAR). Since satisfactory outcomes were obtained from the controlled pollen exposure at the chamber, we conducted preliminary studies in volunteers with SAR. METHODS: Ten volunteers with SAR sensitive to Japanese cedar (JC) pollen were enrolled in this study. In order to investigate the intranasal and intraocular pollen number, volunteers were initially exposed to a low concentration of JC (2500 grains/m3) for at most 1 hour in this chamber. Before and after the exposure, nasal cavities and eyes were washed with 100ml and 25 ml of saline, respectively. Nasal and eye washing solutions were collected and the number of JC pollen was counted. After 3 hours the volunteers were subsequently exposed to a moderate concentration of JC (4500 grains/m3) for 2 hours. Subjective nasal and ocular symptoms were recorded and the amount of nasal secretion was measured during the allergen exposure periods. RESULTS: During the initial exposure, all volunteers except one stayed in the chamber for 1 hour without any nasal or ocular symptoms. The number of pollen in the nose and eyes was 249.2 +/- 120.9 and 13.6 +/- 13.6 grains, respectively. During the subsequent 2-hour exposure to JC pollen, nasal and ocular symptoms developed gradually in a time dependent manner in all the volunteers except one. CONCLUSIONS: This is the first clinical study using Japanese cedar pollen under well-controlled conditions in the OHIO chamber in which the induction of allergic symptoms was observed. The OHIO chamber will be useful for studying allergic rhinitis in Japan.

The detection of red pigment-concentrating hormone (RPCH) in crustacean eyestalk tissues using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry: [M + Na]+ ion formation in dried droplet tissue preparations.

Red pigment-concentrating hormone (RPCH), an octapeptide found in crustaceans and insects with the sequence pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, is an N- and C-terminally blocked uncharged peptide. These structural features are shared with many members of the larger adipokinetic hormone (AKH)/RPCH peptide family in insects. We have applied vacuum UV matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron mass spectrometry (FTMS) to the direct analysis of crustacean sinus gland tissues, using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, and have found that RPCH is detected in the cationized, [M + Na]+, form under conditions where other peptides in the direct tissue spectra are protonated without accompanying [M + Na]+ or [M + K]+ satellite peaks. The [M + H]+ ion for RPCH is not detected in tissue samples or for an RPCH standard, even when care is taken to eliminate metal ions. This behavior is not unprecedented; however, both direct tissue spectra and SORI-CID spectra provide no clues to suggest that the ionizing agent is a metal cation. In this communication, we characterize the MALDI-FTMS ionization and SORI-CID mass spectra of the [M + Na]+ and [M + K]+ ions from RPCH, and report on the detection of this neuropeptide in sinus gland tissues from the lobster Homarus americanus and the kelp crab Pugettia producta. We describe two strategies, an on-probe extraction procedure and a salt-doping approach, that can be applied to previously analyzed MALDI tissue samples to enhance and unmask sodiated peptides that may otherwise be mistaken for novel neuropeptides.

An antibody to recombinant crustacean hyperglycaemic hormone of Nephrops norvegicus cross-reacts with neuroendocrine organs of several taxa of malacostracan Crustacea.

The crustacean hyperglycaemic hormones (cHHs) are multifunctional neuropeptides that play a central role in the physiology of crustaceans. A partial cDNA coding for cHH of the Norway lobster, Nephrops norvegicus, was cloned; this cDNA was fused to glutathione- S-transferase (GST) to obtain a recombinant fusion protein that was used to raise a rabbit antiserum and to perform a biological assay. The specificity of the purified antibody was demonstrated by means of Western blotting. To validate the specificity of the purified antibody to the cHH of N. norvegicus and its cross-reactivity with other species, we performed standard immunocytochemistry of the eyestalk on: (1) paraffin sections of the decapod species N. norvegicus, Munida rugosa and Astacus leptodactylus and of the stomatopod Squilla mantis; (2) semithin resin sections of N. norvegicus and Palaemon elegans; (3) ultrathin sections of N. norvegicus sinus gland (transmission electron microscopy studies). The pattern of immunoreactivity shown by N. norvegicus eyestalk sections conforms to distribution, relative amount and ultrastructural features of cHH-containing neurons and nerve endings as reported in the previous literature. In all the crustacean species examined, the antibody marks precisely the X organ-sinus gland complex and unspecific staining is completely lacking. In addition, its specific cross-reaction by immunoprecipitation depletes shrimp eyestalk extract of hyperglycaemic activity in an in vivo bioassay. The results obtained show a cHH-specific molecular recognition despite the fact that the species tested belong to systematic groups increasingly remote in the phylogenetic tree. The antibody could be used for advancing our knowledge on cHH activity in a variety of crustacean species, e.g. for monitoring reproductive and stress conditions.

Molecular cloning and characterisation of a novel membrane receptor gene from the lobster Jasus edwardsii.

The eyestalk of the lobster, Jasus edwardsii, is an important source for hormones involved in the regulation of growth and reproduction. How these hormones transfer their messages to the cell and nucleus is not known. This paper describes the cloning, characterization and expression analyses of two genes that code for two membrane-associated peptides that may be involved in signal transduction. These genes, peJK2 and peJK3, were isolated from a cDNA library derived from lobster eyestalk mRNAs. The two clones shared 96.6 % sequence homology, and code for putative proteins of 110 and 113 amino acids, respectively. These were likely to be two allelic forms of the same gene. Northern blot analysis using these clones as probes detected the same mRNA from eyestalk, muscle and epithelial extracts, but with greater intensity in the eyestalk extract. In situ hybridisation also indicated the predominant expression of these genes in the eyestalk. Analysis of the putative protein sequences showed that they contained two transmembrane (TM) helices, a short amino acid sequence sharing high homology with the G-protein-coupled receptor (GPCR) motif in the second TM, a signal sequence between the TMs, and a protein kinase phosphorylation site at the C termini. Sequence analyses therefore suggested that the deduced peptides may function in signal transduction.

Biosynthesis and tissue deposition of docosahexaenoic acid (22:6n-3) in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) weighing ca. 5 g and previously acclimated for 8 wk on a diet comprising vegetable oil (11%), fish meal (5%), and casein (48%) as the major constituents were fed a pulse of diet containing deuterated (D5) (17,17,18,18,18)-18:3n-3 ethyl ester. The synthesis and tissue distribution of D5-22:6n-3 was determined 3, 7, 14, 24, and 35 d after the pulse. The whole-body accumulation of D5-22:6n-3 was linear over the first 7 d, corresponding to a rate of 0.54 +/- 0.12 microg D5-22:6n-3/g fish/mg D5-18:3n-3 eaten/d. Maximal accretion of D5-22:6n-3 was 4.3 +/- 1.2 microg/g fish/mg of D5-18:3n-3 eaten after 14 d. The amount of D5-22:6n-3 peaked in liver at day 7, in brain and eyes at day 24, and plateaued after day 14 in visceral and eye socket adipose tissue and in the whole fish. The majority of D5-22:6n-3 was found in the carcass (remaining tissues minus the above tissues analyzed separately) at all times. On a per milligram lipid basis, liver and eyes had the highest concentration of D5-22:6n-3. The experimental diet also contained 21:4n-6 ethyl ester as a marker to estimate the amount of food eaten by individual fish. From such estimates it was calculated that the great majority of the D5-tracer was catabolized, with the combined recovery of D5-18:3n-3 plus D5-22:6n-3 being 2.6%. The recovery of 21:4n-6 was 57.6%. The concentration of 22:6n-3 in the fish decreased during the 13-wk period, and the amount of 22:6n-3 synthesized from 18:3n-3 was only about 5% of that obtained directly from the fish meal in the diet.

Two opsin genes from the vetch aphid, Megoura viciae.

The cDNAs of two opsins (Megopsin1 and Megopsin2) from the vetch aphid, Megoura viciae, have been sequenced and encoded for gene products with 378 and 371 amino acid residues, respectively. Phylogenetic analysis reveals that Megopsin1 falls into the insect long-wavelength opsin group and Megopsin2 is a member of the insect UV-wavelength opsins. Both opsins share the key features of G-protein-coupled receptors and the specific motifs of photopigments. In situ hybridization demonstrated that the transcripts of Megopsin1 and Megopsin2 were expressed in the retinula cells of the compound eyes.

Immune corticotropin-releasing hormone is present in the eyes of and promotes experimental autoimmune uveoretinitis in rodents.

We examined the presence and potential role of local corticotropin-releasing hormone (CRH) in experimental uveitis in rodents. This 41-amino acid peptide, originally isolated from the hypothalamus, is also secreted locally in experimentally induced and natural inflammatory sites, where it exerts autocrine or paracrine proinflammatory effects. Female Lewis rats were immunized with the major pathogenic epitope (R16 peptide) of the interphotoreceptor retinoid-binding protein in complete Freund's adjuvant, monitored daily, and killed 8, 9, 10, 12, 14, or 18 days later, after having developed uveoretinitis. Immunoreactive CRH (IrCRH) was detected by immunohistochemistry in the uveitic eyes in the cytoplasm of inflammatory cells (macrophages, lymphocytes, and polymorphonuclear cells) infiltrating the iris, ciliary body, vitreous, retina, and choroid depending on the stage of the disease. The intensity of the IrCRH staining was positively correlated with the severity of the disease based on morphological criteria. The amount of IrCRH measured by RIA varied between 0.18 +/- 0.03 (mean +/- SE) and 0.79 +/- 0.07 pmol/g wet tissue (8th and 14th day of the disease, respectively). Ophthalmic IrCRH in uveitic rat eyes had similar chromatographic mobility as rat/human CRH-(1-41) by HPLC. Furthermore, female B10.A mice were immunized with interphotoreceptor retinoid-binding protein and treated during the induction (0-7 days) or expression (8-16 days) stages of the disease with ip injections of the anti-CRH antibody TS-2 or placebo nonimmune rabbit serum. The early anti-CRH treatment significantly decreased the disease intensity compared to that in placebo- or late-treated animals (P < 0.05, by analysis of variance). We conclude that IrCRH is present at the site of inflammation in rodent experimental uveitis and that its expression correlates with the natural history and intensity of the disease. Immune CRH appears to play an early pathogenetic role in the induction of experimental uveitis.

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.

Cloning and expression of mRNA encoding prepro-gonad-inhibiting hormone (GIH) in the lobster Homarus americanus.

The gonad-inhibiting hormone (GIH) is produced in the eyestalk X-organ sinus gland complex of male and female lobsters, and plays a prominent role in the regulation of reproduction, e.g. inhibition of vitellogenesis in female animals. To study this neurohormone at the mRNA level, we cloned and sequenced a cDNA which encodes GIH in the lobster Homarus americanus. The structure of preproGIH consists of a signal peptide and the GIH peptide itself. A comparative analysis revealed that lobster GIH, together with crab molt-inhibiting hormone, belongs to a separate group of the crustacean hyperglycemic hormone (CHH) peptide family which seems to be unique for crustaceans. Expression studies showed that GIH mRNA is expressed in the eyestalk, indicating that the neuroendocrine center in this optic structure is the only source of GIH. As this center modulates the other (neuro)endocrine organs in crustaceans, it is postulated that GIH regulates production and release of hormones involved in reproduction/molting processes.