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N-methyl-N-nitrosourea (MNU) is known to cause apoptosis of photoreceptor cells and changes in retinal pigment epithelium (RPE). However, the changes in choriocapillaris, which nourishes photoreceptor cells by diffusing tissue fluid through RPE, have not been reported in detail. Therefore, we studied the ultrastructural transformation in and around the choriocapillaris to characterize the interdependence between choriocapillaris and surrounding tissue components in a mouse model. Seven-week-old male C57BL/6 mice were given a single intraperitoneal injection of MNU (60 mg/kg of body weight). Perfusion-fixed eyeballs were examined chronologically using immunohistochemistry and electron microscopy until the photoreceptor cells were lost. Sequential ultrastructural changes were observed in photoreceptor cells, RPE, Bruch's membrane, choriocapillaris, and choroidal melanocytes after an MNU injection. The lumens of the choriocapillaris narrowed following dilation, and the vascular endothelium showed structural alterations. When the photoreceptor cells were completely lost, the choriocapillaris appeared to be in a recovery process. Our results suggest that transport abnormality through Bruch's membrane and structural changes in the choroid might have influenced the morphology of choriocapillaris. The thin wall of the choriocapillaris appears to be the cause of the vulnerability with its altered morphology.

This report presents a case of deep peroneal nerve palsy caused by an injury during drilling for distal locking screws of a T‐shaped locking plate used for osteosynthesis of medial open‐wedge high tibial osteotomy. A cadaveric simulation study reproduced the risk of injury during the surgery., Surgeons should acknowledge the hidden risk of injury of the deep peroneal nerve when drilling for the distal locking screws during medial open‐wedge high tibial osteotomy and avoid drilling too deeply after penetrating the lateral cortex or consider a monocortical fixation fashion.

Herein we describe the practical synthesis of a potent glucokinase activator (1) that has a chiral thiophenyl-pyrrolidine scaffold. The key to the successful synthesis was the application of a telescoped chiral-pool synthesis from a commercially available l-proline methyl ester derivative to introduce the chirality of the thiophenyl-pyrrolidine moiety. This second-generation synthesis of 1 provided several advantages over the previous method including an operational simplicity and avoidance of purification by column chromatography. The industrial relevance of this synthetic method in large-scale preparation was demonstrated by the production of 54.6 kg of 1 with an excellent chemical and optical purity.

Müller glia, the principal glial cell type in the retina, have the potential to reenter the cell cycle after retinal injury. In mammals, proliferation of Müller glia is followed by gliosis, but not regeneration of neurons. Retinal injury is also accompanied by phagocytic removal of degenerated cells. We here investigated the possibility that proliferation and gliosis of Müller glia and phagocytosis of degenerated cells may be regulated by the same molecular pathways. After N-methyl-N–nitrosourea-induced retinal injury, degenerated photoreceptors were eliminated prior to the infiltration of microglia/macrophages into the outer nuclear layer, almost in parallel with cell cycle reentry of Müller glia. Inhibition of microglia/macrophage activation with minocycline did not affect the photoreceptor clearance. Accumulation of lysosomes and rhodopsin-positive photoreceptor debris within the cytoplasm of Müller glia indicated that Müller glia phagocytosed most photoreceptor debris. Pharmacological inhibition of phosphatidylserine and Rac1, key regulators of the phagocytic pathway, prevented cell cycle reentry, migration, upregulation of glial fibrillary acidic protein, and phagocytic activity of Müller glia. These data provide evidence that phosphatidylserine and Rac1 may contribute to the crosstalk between different signaling pathways activated in Müller glia after injury.

The anatomical variations of the confluence of sinuses were examined, focusing on the continuity of the superior sagittal sinus (SSS) and the transverse sinuses (TSs). In the 142 specimens studied, there were 72 symmetric cases (50.7%) and 70 asymmetric cases (49.3%). The symmetric group (no dominant type) was categorized into 34 cases of bifurcation (23.9%) and 38 cases of confluence (26.8%). The asymmetric group was categorized into 54 cases of the right-dominant type (38.0%) and 16 cases of the left-dominant type (11.3%). The right-dominant type was further categorized into 38 partially-communicating (26.8%) and 16 non-communicating types (11.3%). The left-dominant type was categorized into 11 partially-communicating (7.7%) and 5 non-communicating types (3.5%). In summary, the SSS asymmetrically drained into one TS in about half of the cases studied. The right-dominant type was about three to four times as common as the left-dominant type. The draining pattern shown by the asymmetric group could provoke intracranial hypertension due to unilateral jugular vein obstruction. In order to avoid this risk in cases of neck dissection, jugular vein catheterization, or hypercoagulopathy, preoperative evaluations of the dural sinus variations via MR venography, three-dimensional CT, or plain X-ray of the skull are recommended.

Background Cyclin-dependent kinase (CDK) inhibitors play an important role in regulating cell cycle progression, cell cycle exit and cell differentiation. p27KIP1 (p27), one of the major CDK inhibitors in the retina, has been shown to control the timing of cell cycle exit of retinal progenitors. However, the precise role of this protein in retinal development remains largely unexplored. We thus analyzed p27-deficient mice to characterize the effects of p27 loss on proliferation, differentiation, and survival of retinal cells. Methods Expression of p27 in the developing and mature mouse retina was analyzed by immunohistochemistry using antibodies against p27 and cell type-specific markers. Cell proliferation and differentiation were examined in the wild-type and p27-deficient retinas by immunohistochemistry using various cell cycle and differentiation markers. Results All postmitotic retinal cell types expressed p27 in the mouse retinas. p27 loss caused extension of the period of proliferation in the developing retinas. This extra proliferation was mainly due to ectopic cell cycle reentry of differentiating cells including bipolar cells, Müller glial cells and cones, rather than persistent division of progenitors as previously suggested. Aberrant cell cycle activity of cones was followed by cone death resulting in a significant reduction in cone number in the mature p27-deficient retinas. Conclusions Although expressed in all retinal cell types, p27 is required to maintain the quiescence of specific cell types including bipolar cells, Müller glia, and cones while it is dispensable for preventing cell cycle reentry in other cell types.

To obtain an efficient and practical route to a novel glucokinase activator, we investigated a novel synthetic method for the preparation of its key intermediate (S)-5-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-2-methylthiophene-3-carboxylic acid through the asymmetric transfer hydrogenation of a pyrroline derivative. The hydrogenation of this pyrroline derivative using the iridium (III)-prolinamide complex Cp*IrCl[(R)-PA] at atmospheric pressure provided an initial intermediate in approximately 50% ee. Further purification via recrystallization provided the desired key intermediate in an excellent enantiopurity, which was applicable to practical use.

Glucokinase (GK) is an enzyme that plays an important role as a glucose sensor while maintaining whole body glucose homeostasis. Allosteric activators of GK (GKAs) have the potential to treat type 2 diabetes mellitus. To identify novel GKAs, a series of compounds based on a thiophenyl-pyrrolidine scaffold were designed and synthesized. In this series, compound 38 was found to inhibit glucose excursion in an oral glucose tolerance test (OGTT) in mice. Optimization of 38 using a zwitterion approach led to the identification of the novel GKA 59. GKA 59 exhibited potent blood glucose control in the OGTT test as well as a favorable safety profile. Owing to low pancreatic distribution, compound 59 primarily activates GK in the liver. This characteristic could overcome limitations of other GKAs, such as hypoglycemia, increased plasma triglycerides, and loss of efficacy.

The clinical anatomy of the recurrent artery of Heubner (RAH) was examined, focusing on its number, origin, and course, in a large number of brain specimens. We studied 724 RAH in total from 357 brain specimens (714 hemispheres). In 98.74 % of 714 cases there were one or more RAHs, while it was absent in 1.26 % of cases. There was a single RAH in 96.22 % of cases, double in 2.38 % of cases, and triple in 0.14 % of cases. In this study, three origin types of the RAH were defined. We defined A1 and A2 segment of the anterior cerebral artery (ACA) as the artery from the origin of the ACA to the junction of the anterior communicating artery (AComA) and the artery from the junction of the AComA to the anterior border of the corpus callosum, respectively. In 76.2 % of 724 arteries, the RAH originated from the junction of the A1 and A2 segment of the ACA. In 16.3 %, the RAH originated from the A2 segment of the ACA. In 7.5 %, the RAH originated from the A1 segment of the ACA. The course of the RAH was superior to the A1 segment of the ACA in 30.1 % of 724 arteries, anterior in 62.2 %, and posterior in 7.7 %. It is of great importance for neurosurgeons to understand the detailed anatomical variations of the RAH before operating to prevent operative complications resulting in neurological deficits.

Rapid saltatory nerve conduction is facilitated by myelin structure, which is produced by Schwann cells (SC) in the peripheral nervous system (PNS). Proper development and degeneration/regeneration after injury requires regulated phenotypic changes of SC. We have previously shown that glutamate can induce SC proliferation in culture. Here we show that glutamate signals through metabotropic glutamate receptor 2 (mGluR2) to induce Erk phosphorylation in SC. mGluR2-elicited Erk phosphorylation requires ErbB2/3 receptor tyrosine kinase phosphorylation to limit the signaling cascade that promotes phosphorylation of Erk, but not Akt. We found that Gβγ and Src are involved in subcellular signaling downstream of mGluR2. We also found that glutamate can transform myelinating SC to proliferating SC, while inhibition of mGluR2 signaling can inhibit demyelination of injured nerves in vivo. These data suggest pathophysiological significance of mGluR2 signaling in PNS and its possible therapeutic importance to combat demyelinating disorders including Charcot-Marie-Tooth disease.

Color vision is supported by retinal cone photoreceptors that, in most mammals, express two photopigments sensitive to short (S-opsin) or middle (M-opsin) wavelengths. Expression of the Opn1sw and Opn1mw genes, encoding S-opsin and M-opsin, respectively, is under the control of nuclear receptors, including thyroid hormone receptor beta2 (TRbeta2), retinoid X receptor gamma (RXRgamma), and RORbeta, a member of the retinoic acid receptor-related orphan receptor (ROR) family. We now demonstrate that RORalpha, another member of the ROR family, regulates Opn1sw, Opn1mw, as well as Arr3 (cone arrestin) in the mouse retina. RORalpha expression is detected in cones by postnatal day 3 and maintained through adulthood. The retinas of staggerer mice, carrying a null mutation of RORalpha, show significant down-regulation of Opn1sw, Opn1mw, and Arr3. RORalpha acts in synergy with cone-rod homeobox transcription factor (Crx), to activate the Opn1sw promoter in vitro. Chromatin immunoprecipitation assays reveal that RORalpha directly binds to the Opn1sw promoter, Opn1mw locus control region, and the Arr3 promoter in vivo. Our data suggest that RORalpha plays a crucial role in cone development by directly regulating multiple cone genes.

Brain-derived neurotrophic factor (BDNF) regulates many aspects of neuronal development, including survival, axonal and dendritic growth and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF in retinal development, the retinal cell types expressing BDNF remains poorly defined. The goal of the present study was to determine the localization of BDNF in the mammalian retina, with special focus on the subtypes of amacrine cells, and to characterize, at the cellular level, the effects of constant light exposure during early postnatal period on retinal expression of BDNF. Retinas from 3-week-old rats reared in a normal light cycle or constant light were subjected to double immunofluorescence staining using antibodies to BDNF and retinal cell markers. BDNF immunoreactivity was localized to ganglion cells, cholinergic amacrine cells and dopaminergic amacrine cells, but not to AII amacrine cells regardless of rearing conditions. Approximately 75% of BDNF-positive cells in the inner nuclear layer were cholinergic amacrine cells in animals reared in a normal lighting condition. While BDNF immunoreactivity in ganglion cells and cholinergic amacrine cells was significantly increased by constant light rearing, which in dopaminergic amacrine cells was apparently unaltered. The overall structure of the retina and the density of ganglion cells, cholinergic amacrine cells and AII amacrine cells were unaffected by rearing conditions, whereas the density of dopaminergic amacrine cells was significantly increased by constant light rearing. The present results indicate that cholinergic amacrine cells are the primary source of BDNF in the inner nuclear layer of the rat retina and provide the first evidence that cholinergic amacrine cells may be involved in the visual activity-dependent regulation of retinal development through the production of BDNF. The present data also suggest that the production or survival of dopaminergic amacrine cells is regulated by early visual experience.

To find novel PPARdelta-selective agonists, we designed and synthesized phenylpropanoic acid derivatives bearing 6-substituted benzothiazoles. Optimization of this series led to the identification of a potent and selective PPARdelta agonist 17. Molecular modeling suggested that compound 17 occupies the Y-shaped pocket of PPARdelta appropriately.

To develop novel PPARalpha ligands, we designed and synthesized several 3-{3-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic acid derivatives. Compound 10, the meta isomer of a PPARgamma agonist 1, has been identified as a PPARalpha ligand. The introduction of methyl and ethyl groups at the C-2 position of the propanoic acid of 10 further improved the PPARalpha-binding potency.

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.

To develop novel PPARγ ligands, we synthesized thirteen 3-{4-(2-aminoethoxy)phenyl}propanoic acid derivatives, which are designed based on the structures of rosiglitazone and 15d-PGJ2. Among these compounds, compound 9 was found to be as potent as rosiglitazone in a binding assay and a preadipocyte differentiation test. Molecular modeling suggested that the nonyl group of 9 interacted with hydrophobic amino acid residues constructing the hydrophobic region of PPARγ protein where the alkyl chain of 15d-PGJ2 is expected to be located.

Systematic investigation of the varying size of the alkoxy moiety of the lithium ester enolates in the chiral ligand mediated reaction with imines led to the improvement of the enantioselectivity of β-lactam products in up to 93%. The asymmetric induction step was indicated to be the addition step, not the cyclization step.

The fate of neutrophils in the peripheral circulation is poorly understood. In this study, the role of Kupffer cells in eliminating aged and apoptotic neutrophils was investigated. Liver, spleen, lung, and blood samples from Wistar rats were examined by light and electron microscopy, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method, and immunohistochemistry after the intravenous injection of OK-432, a streptococcal preparation. Neutrophils were trapped predominantly in the periportal and midzonal regions of hepatic lobules and were in contact with endothelial cells and Kupffer cells, or were surrounded by Kupffer cells. The trapping of neutrophils peaked after 6 hours. Apoptotic neutrophils, with or without buds, were found in the lumen of hepatic sinusoids as early as 6 hours, reached maximal levels after 12 hours, and represented greater than 60% of the total number of neutrophils in the liver. The presence of apoptotic neutrophils was correlated with the degree of neutrophil phagocytosis. Double-staining showed that TUNEL-positive neutrophils were phagocytosed or encircled by ED1- or ED2-positive Kupffer cells. In contrast, apoptosis and phagocytosis of neutrophils were rare in the spleen, lung, and peripheral blood. These results suggested that the appearance of apoptotic neutrophils in the hepatic sinusoids and their rapid clearance by Kupffer cells occurs after the invasion of bacteria (i.e., bacteremia or bacteriotoxemia) or the release of inflammatory mediators into the blood stream. These findings have important implications for the regulation of neutrophil homeostasis, the limitation of inflammation and tissue injury, and provide insight into the physiological removal of circulating, senescent neutrophils.

PURPOSE Muller glia, the principal glial cell type in the retina, have the potential to proliferate and regenerate neurons after retinal damage. However, unlike the situation in fish and birds, this capacity of Muller glia is extremely limited in mammals. To gain new insights into the mechanisms that hamper retinal regeneration in mammals, we examined the cell cycle progression and DNA damage response in Muller glia after retinal damage. METHODS Expression of cell cycle-related proteins and DNA damage response were analyzed in adult rat and mouse retinas after N-methyl-N-nitrosourea (MNU)- or N-methyl-D-aspartate (NMDA)-induced retinal damage. Zebrafish and postnatal rat retinas were also investigated for comparison. Analysis was conducted by using immunofluorescence, Western blotting, and quantitative real-time polymerase chain reaction. RESULTS In the rat retina, most Muller glia reentered the cell cycle after MNU-induced photoreceptor damage while no proliferative response was observed in the mouse model. Cell cycle reentry of rat Muller glia was accompanied by DNA damage response including the phosphorylation of the histone variant H2AX and upregulation of p53 and p21. The DNA damage response was also observed in rat Muller glia after NMDA-induced loss of inner retinal neurons, but not in zebrafish Muller glia or rat retinal progenitor cells. CONCLUSIONS Our findings suggest that the DNA damage response induced by unscheduled cell cycle reentry may be one of the mechanisms that limit the proliferative and regenerative capacity of Muller glia in the mammalian retina.

We disclosed a novel series of G-protein coupled receptor 119 (GPR119) agonists based on a bicyclic amine scaffold. Through the optimization of hit compound 1, we discovered that the basic nitrogen atom of bicyclic amine played an important role in GPR119 agonist activity expression and that an indanone in various bicyclic rings was suitable in this series of compounds. The indanone derivative 2 showed the effect of plasma glucose control in oGTT and scGTT in the rodent model.

The reactions of 3-acyl-2-azabicyclo[2.2.1]hept-5-enes with phenyl isocyanate, phenyl isothiocyanate, and ethoxycarbonyl isothiocyanate followed by cyclization reactions under basic conditions respectively provided a hydantoin, a thiohydantoin, and a 1,3-dihydro-2H-imidazole-2-thione incorporated with the azabicyclic skeleton. Treatment of the imidazolethione with Raney nickel gave a phenyl-substituted imidazole fused with 2-azabicyclo[2.2.1]hept-2-ene. The effect of annulation on the 13C NMR of the imidazole ring is discussed.

The formal [3+2] cycloaddition of 5-methoxy-2-(p-methoxyphenyl)oxazole with 2(S)-(benzyloxy)-propanal and 2(S)- [(tert-butyldimethylsilyl)- oxy]propanal in the presence of tin(IV) chloride gave cis-(4R,5S,1'S)- and trans-(4S,5S,1'S)-5-(1'-alkoxyethyl)-4-(methoxycarbonyl)-2-(p-me- thoxyphenyl)-2-oxazolines with high diastereoselectivity (94 and 92% selectivity, respectively). A similar reaction of 5-methoxy-2-(p-metho- xyphenyl)oxazole with 2,3-di-O-benzyl-D-glyceraldehyde gave methyl cis- (4S,5R,1'R)-2-(p-methoxyphenyl)-5-[1',2'-bis(benzyloxy)ethyl]-2-oxazo- line-4-carboxylate with high diastereoselectivity (>95% selectivity), and the latter was easily converted to biologically important chiral 2-amino-1,3,4,5-tetrol derivatives

The reaction of 2-alkyl- or 2-aryl-substituted 5-methoxy-4-(p-nitrophenyl)oxazoles with tetracyanoethylene gave methyl esters of 5-substituted 3,3,4,4-tetracyano-2-(p-nitrophenyl)-3,4-dihydro-2H-pyrrole-2-carboxylic acids as formal [3 + 2] cycloadducts through oxazole ring opening. Zwitterionic mechanisms were proposed on the basis of the solvent effect on the reaction. The molecular structure of 2-isopropyl-substituted product was determined by means of X-ray crystallography. Crystals of the cycloadduct are orthorhombic with space group Pcab having unit-cell dimensions of a = 17.998 (2), b = 27.188 (6), c = 15.833 (3) A, and Z = 16, which contains two crystallographically independent molecules. The final R value is 0.070 for 4629 observed reflections.

The reactions of 3-acyl-2-azabicyclo[2.2.1]hept-5-enes with phenyl isocyanate, phenyl isothiocyanate, and ethoxycarbonyl isothiocyanate followed by cyclization reactions under basic conditions respectively provided a hydantoin, a thiohydantoin, and a 1,3-dihydro-2H-imidazole-2-thione incorporated with the azabicyclic skeleton. Treatment of the imidazolethione with Raney nickel gave a phenyl-substituted imidazole fused with 2-azabicyclo[2.2.1]hept-2-ene. The effect of annulation on the 13C NMR of the imidazole ring is discussed.

Systematic investigation of the varying size of the alkoxy moiety of the lithium ester enolates in the chiral ligand mediated reaction with imines led to the improvement of the enantioselectivity of β-lactam products in up to 93%. The asymmetric induction step was indicated to be the addition step, not the cyclization step.

The reaction of 2-alkyl- or 2-aryl-substituted 5-methoxy-4-(p-nitrophenyl)oxazoles with tetracyanoethylene gave methyl esters of 5-substituted 3,3,4,4-tetracyano-2-(p-nitrophenyl)-3,4-dihydro-2H-pyrrole-2-carboxylic acids as formal [3 + 2] cycloadducts through oxazole ring opening. Zwitterionic mechanisms were proposed on the basis of the solvent effect on the reaction. The molecular structure of 2-isopropyl-substituted product was determined by means of X-ray crystallography. Crystals of the cycloadduct are orthorhombic with space group Pcab having unit-cell dimensions of a = 17.998 (2), b = 27.188 (6), c = 15.833 (3) A, and Z = 16, which contains two crystallographically independent molecules. The final R value is 0.070 for 4629 observed reflections.

The [3 + 2] cycloaddition of 5-alkoxyoxazoles with aldehydes in the presence of the organoaluminum reagent, which was prepared from (±)-2,2′-dihydroxy-1,1′-dinaphthyl and AlMe3, gave cis-4-alkoxycarbonyl-2-oxazolines in high regio- and stereoselective manner.

Hydroxyindole O-methyltransferase (HIOMT)-immunoreactive cells and melatonin synthesis were demonstrated in bovine epithalamus (including the pineal gland) with monoclonal antibodies and cRNA probes to HIOMT. The HIOMT-immunoreactive product was present in the cytoplasm of pinealocytes. All identifiable pinealocytes were clearly labeled in the pineal gland. The expression of the HIOMT gene was first identified in pinealocyte cytoplasm by in-situ hybridization (ISH). The distribution of the hybridization-positive cells in the pineal gland was compatible with that revealed by immunocytochemistry using the monoclonal antibody to HIOMT. In addition, HIOMT transcripts were found in the medial habenular nucleus, and the habenular and posterior commissure; they may correspond to S-antigen-immunoreactive cells demonstrated in the same regions of the hamster and the mouse. In these regions, the hybridization-positive cells did not exhibit HIOMT-immunoreactivity; thus, cells devoid of immunoreactivity may synthesize but rapidly transport the newly synthesized proteins. These results indicate (1) that the conversion of N-acetylserotonin into melatonin takes place in the cytoplasm of pinealocytes, (2) that some epithalamic cells in the habenular area may synthesize melatonin, and (3) that melatonin may act as a chemical messenger in centrally directed processes, as shown by using S-antigen immunocytochemistry.

A weak, nonselective G protein-coupled receptor 120 (GPR120) agonist 10 was found by screening a series of carboxylic acids derived from the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist 3. Modification based on the homology model of GPR120 led to the first GPR120-selective agonist 12. These results provide a basis for constructing new tools for probing the biology of GPR120 and for developing new candidate therapeutic agents.

To find novel PPAR ligands, we prepared several 3-{3 or 4-[2-(nonylpyridin-2-ylamino)ethoxy]phenyl}propanoic acid derivatives which were designed based on the structure of our previous PPARgamma ligand 1. In PPAR binding affinity assays, compound 4, which had an ethoxy group at the C-2 position of the propanoic acid of 1, showed selective binding affinity for PPARgamma. Compound 3, with an ethyl group at the C-2 position, was found to be a PPARalpha/gamma dual ligand. Compound 6, the meta isomer of 1, has been shown to be a PPARalpha ligand. The introduction of methyl (7) and ethyl (8) groups to the C-2 position of the propanoic acid of 6 further improved PPARalpha-binding potency. In cell-based transactivation assay, compounds 3 and 4 showed dual-agonist activity toward PPARalpha and PPARgamma. Compound 6 was found to be a triple agonist and compound 8 proved to be a selective PPARalpha agonist. In the human hypodermic preadipocyte differentiation test, it was demonstrated that the maximal activity of compounds 3 and 4 was higher than that of rosiglitazone.

AII amacrine cells are critical interneurons in the rod pathway of mammalian retina, active primarily in dim lighting conditions. Melatonin, a neuromodulator produced at night in the retina, is believed to induce retinal adaptation to dim lighting conditions in most vertebrate species examined to date, including humans. We hypothesized that melatonin may influence retinal light adaptation by acting on AII cells directly and thus investigated whether melatonin receptors were expressed in AII neurons. Postmortem nonpathological eyes from four human donors as well as two eyes from two Macaque Fasicularis monkeys were analyzed. Double immunocytochemistry was performed using an anti-MT 1 antibody and an antibody to calretinin, an AII marker. Analysis utilized confocal microscopy. A polyclonal anti-calretinin antibody labelled amacrine cells exhibiting the distinct AII morphology, in both human and macaque retina. MT 1 immunoreactivity in macaque retina was similar to human staining, in that horizontal, amacrine and ganglion cell bodies were stained, as were inner segments of photoreceptors. In human retina 86% of calretinin positive cells expressed the MT 1 receptor peripherally, whereas centrally, 78% colocalization was observed. In the macaque retina, 100% of AII amacrine cells expressed MT 1 immunoreactivity both centrally and peripherally. That virtually all AII neurons express the MT 1 receptor in both human and macaque retina, may provide the first evidence demonstrating a role for melatonin in AII regulation, furthering the hypothesis of melatonin function in retinal light adaptation.

Melatonin's function in human vision is far from understood, in part because of the lack of information on its cellular targets. Therefore, expression and localization of the MT(1) melatonin receptor in human retina was examined.Postmortem nonpathologic human eyes from nine donors were investigated, three by reverse transcription-polymerase chain reaction (RT-PCR) for MT(1) and MT(2) transcripts and six by immunocytochemistry, using a peptide-specific anti-MT(1) receptor antibody.RT-PCR suggested that both MT(1) and MT(2) transcripts had similar levels of expression. Vertical slices of human retina demonstrated MT(1) immunoreactivity in cell bodies along the outer border of the inner nuclear layer (INL), along the inner border of the INL, in cell bodies within the ganglion cell layer (GCL), and in the inner segments (IS) of photoreceptors. Double immunolabeling using anti-MT(1) and tyrosine hydroxylase revealed that 69% of CA1 and 63% of CA2 dopaminergic neurons exhibited MT(1) immunoreaction. Double immunolabeling with anti-parvalbumin, a horizontal cell marker, showed that MT(1)-positive cells along the outer INL border were exclusively horizontal cells, and that 18% of horizontal cells in central retina expressed MT(1). Double staining with MT(1) and markers for both rod and cone photoreceptors suggest that IS staining is present on rod cells.The MT(1) receptor is expressed in diverse neuronal cell types in the human retina, providing evidence of a significant role for melatonin and this receptor subtype in human vision.

Distribution of the mt1 melatonin receptor in the guinea pig retina was immunocytochemically investigated using peptide-specific anti-mt1 receptor antibody. Western blots of the guinea pig retina showed a single band at approximately 37 kilodalton (kD) immunoreactive to the anti-mt1 antibody. The most intense immunoreactivity for the mt1 receptor was detected in the cell bodies of ganglion cells. Their dendrites and axons were also immunolabeled. Subpopulations of amacrine cells, the inner plexiform layer, and the outer plexiform layer also exhibited moderate to weak immunolabeling. The mt1-positive amacrine cells were located either at the vitreal border of the inner nuclear layer or displaced in the ganglion cell layer. Double immunolabeling using antibodies to the mt1 receptor and tyrosine hydroxylase revealed that the majority of dopaminergic amacrine cells showed mt1 immunoreactivity. Almost all the 1CA type dopaminergic cells were mt1 positive while the 2CA type cells less frequently exhibited mt1 immunoreaction. By double immunolabeling for the mt1 receptor and GABA, more than 50% of the mt1-immunoreactive amacrine cells were shown to be GABAergic neurons. Approximately one-third of the GABAergic amacrine cells were immunolabeled for the mt1 receptor. The present results demonstrate expression of the mt1 receptor in diverse neuronal cell types in the guinea pig retina and provide the first evidence for the direct effect of melatonin on dopaminergic and GABAergic amacrine cells via the mt1 receptor.

Melatonin is synthesized in the retina at night and acts as a local modulator within this tissue by mediating the effects of darkness. We investigated the expression and localization of the mt1 (Mel1a) melatonin receptor in rat retina in order to disclose the cellular and molecular bases of melatonin's action in the mammalian retina. Western blotting of the mt1 receptor in rat retina exhibited a single immunoreactive band of approximately 37,000 mol. wt, which corresponds to the predicted molecular size of the receptor. The mt1 receptor was immunocytochemically localized to both the inner and outer plexiform layers. During postnatal development, retina from two-week-old rats showed the highest mt1 immunoreactivity; the outer plexiform layer and horizontal cell bodies were strongly immunolabeled, with weaker labeling in the inner plexiform layer. Expression of mt1 receptor messenger RNA in the rat retina was demonstrated by reverse transcription-polymerase chain reaction and in situ hybridization. mt1 receptor transcripts were localized to ganglion cells, amacrine cells and horizontal cells. These results suggest that melatonin influences retinal physiology by acting on multiple retinal cell types, including ganglion, amacrine and horizontal cells, via the mt1 receptor expressed in their processes.

Phylogenetically, the pineal organ changes from a photoreceptor organ endowed with true photoreceptor cells to an endocrine gland influenced by light stimuli perceived in the retina. In poikilothermic vertebrates, the pineal complex contains photoreceptor cells which are synaptically connected to intrapineal second-order neurons. The axons of these second-order neurons constitute the central pinealofugal innervation which connects the pineal complex with various nuclei located in the diencephalon and mesencephalon. Changes in the structure and function of pineal photoreceptors are accompanied by the reduction of intrapineal neurons in the course of evolution. In mammals, pinealocytes receive information from a biological clock system—present inside the suprachiasmatic nucleus of the hypothalamus—entrained by the photoperiod and have lost structural and functional characteristics of photoreceptor cells. Nevertheless, mammalian pinealocytes are considered as derivatives of the pineal photoreceptor cells of anamniotic vertebrates (Oksche, 1971; Collin, 1971). This functional shift of the pinealocyte has been confirmed by immunohistochemical and biochemical studies showing that neuroendocrine pinealocytes of mammals express photoreceptor-specific molecules which otherwise are synthesized only by true (retinal and pineal) photoreceptor cells (Korf et al, 1986; Kramm et al., 1993). Concomitantly with the loss of the direct photosensitivity of pinealocytes, the central pinealofugal innervation is reduced in mammals. Alternatively, the pineal gland of mammals receives a conspicuous autonomic innervation (Cajal, 1904; Kappers, 1960), which is essential for the regulation of melatonin metabolism in these species. The autonomic nervous system also innervates the pineal organ of reptiles and birds, but in these species it appears to be less important than in mammals. Autonomic nerve fibers are scarce or even absent in the pineal organ of poikilothermic vertebrates such as fishes and amphibians (see Vollrath, 1981 for details).

Epithelial remodeling in the rat pineal during fetal development was immunohistochemically analyzed by using antibodies for laminin and cadherin as molecular markers of basal lamina and intercellular junctions, respectively. The proliferation and differentiation of pinealocytes were also investigated in relation to the advance of epithelial remodeling. The pineal anlage of embryonic day 16 is completely covered by basal lamina immunolabeled for laminin. After embryonic day 17, local dissolution of the basal lamina occurs on the epithelial folds, which develop predominantly in the rostral pineal wall. Some pineal cells migrate through these interruptions and form cellular aggregations outside the basal lamina. Cadherin immunostaining reveals focal dissolution of intercellular junctions in epithelial regions protruding into the pineal lumen. Dissolution of the basal lamina and intercellular junctions accompanied by cellular migration into the stromal tissue or into the pineal lumen continues until birth. The distribution of mitotic cells immunolabeled for BrdU is homogeneous throughout the organ during the fetal period, whereas that of differentiating pinealocytes immunoreactive for synaptophysin shows striking regional heterogeneity in close correlation with the remodeling of the pineal epithelium. The migrating cell populations located either outside the basal lamina or inside the pineal lumen are more liable to become synaptophysin-positive than the rest of the epithelium. These results suggest that epithelial remodeling in the fetal pineal is induced, at least in part, by epithelial infolding and that this remodeling promotes the differentiation of pinealocytes.

Cells expressing major histocompatibility complex (MHC) class II (Ia) antigen have been examined during the development of rat pineals and in the pineal gland of adult rats treated with carbon tetrachloride. Cells positive for MHC class II are first detected in the pineal gland of the 7-day-old rat. These positive cells increase in number gradually during development, MHC class II immunoreactivity reaching adult levels at 4 weeks after birth. The MHC class II antigen is intensely labeled on the cell surface, and labeled cells are distributed throughout the organ, several positive cells being gathered into groups. The positive cells are small (7-12 microm in diameter), irregular in shape, and frequently exhibit one or more processes. At the electron-microscopic level, the cytoplasm of positive cells contains few organelles, variously sized empty vacuoles, and a few electron-dense lysosome-like structures. Pinealocytes with synaptic ribbons have been found adjacent to immunoreactive cells. Double-immunoperoxidase staining for MRC OX6, MRC OX42, and ED1 results in OX6(-)/ED1(+)/OX42(+), OX6(-)/ED1(-)/OX42(+), and OX6(+)/ED1(-)/OX42(- )cells. These findings suggest that OX6-positive cells in the pineal can be considered as peripheral dendritic cells. The number of cells expressing MHC class II (Ia) antigen significantly increases in the pineal gland of rats after treatment with carbon tetrachloride (P0.005). Our results indicate that at least some of the OX6-positive cells migrate into the gland from the circulation under these conditions.

In the present study, we demonstrate a cortical and medullary arrangement of parenchymal cells in the bovine pineal gland by using antibodies for neuron-specific enolase, synaptophysin, and hydroxyindole O-methyltransferase (HIOMT) as markers of pinealocytes, and glial fibrillary acidic protein (GFAP) as a marker of interstitial (glial) cells. Furthermore, by means of probes specific for HIOMT mRNA, we have examined possible differences in melatonin synthesis between the cortex and the medulla. Immunoreactive pinealocytes for each antigen investigated are more densely distributed in the cortex than in the medulla. In the cortex, GFAP-positive interstitial cells have large intenselystained somata endowed with several long, thin cytoplasmic processes, whereas in the medulla, they display smaller, less intensely labeled perikarya from which numerous fine short processes emerge. Golgi staining has confirmed these morphological differences between the interstitial cells in the cortex and those in the medulla. An analysis using confocal laser microscopy together with in situ hybridization for HIOMT mRNA has shown that the expression of mRNA transcripts in the cortex is more intense than that in the medulla. The expression of the HIOMT gene in a cluster of cells in the medial habenular nucleus is lower than that in pinealocytes of the pineal organ proper.

Tissue kallikrein in the rat pineal gland was immunocytochemically investigated with the aid of specific antiserum against rat urinary kallikrein. We also compared the tissue kallikrein immunoreactivity of the pineal gland with that of the submandibular gland and kidney, which have been well established as tissue kallikrein-synthesizing organs. The cytoplasm of pinealocytes from both the superficial and the deep portion of the gland exhibited specific immunolabeling for tissue kallikrein, but the immunoreaction was weaker than that observed in exocrine organs. Two types of tissue kallikrein-immunoreactive pinealocytes were distinguished; the first predominant type displayed moderate immunostaining, whereas a small number of cells, the second type, were so intensely labeled that their cytoplasmic processes were clearly outlined. The results of the present study suggest the existence of different types of pinealocytes and a potential physiological role of tissue kallikrein in the rat pineal gland.