Physiological and pathophysiological roles of acidic mammalian chitinase (CHIA) in multiple organs.

Acidic mammalian chitinase (CHIA) belongs to the 18-glycosidase family and is expressed in epithelial cells and certain immune cells (such as neutrophils and macrophages) in various organs. Under physiological conditions, as a hydrolase, CHIA can degrade chitin-containing pathogens, participate in Type 2 helper T (Th2)-mediated inflammation, and enhance innate and adaptive immunity to pathogen invasion. Under pathological conditions, such as rhinitis, ocular conjunctivitis, asthma, chronic atrophic gastritis, type 2 diabetes, and pulmonary interstitial fibrosis, CHIA expression is significantly changed. In addition, studies have shown that CHIA has an anti-apoptotic effect, promotes epithelial cell proliferation and maintains organ integrity, and these effects are not related to chitinase degradation. CHIA can also be used as a biomolecular marker in diseases such as chronic atrophic gastritis, dry eye, and acute kidney damage caused by sepsis. Analysis of the authoritative TCGA database shows that CHIA expression in gastric adenocarcinoma, liver cancer, renal clear cell carcinoma and other tumors is significantly downregulated compared with that in normal tissues, but the specific mechanism is unclear. This review is based on all surveys conducted to date and summarizes the expression patterns and functional diversity of CHIA in various organs. Understanding the physiological and pathophysiological relevance of CHIA in multiple organs opens new possibilities for disease treatment.

Comparative Proteomics Analysis of the Postmitochondrial Supernatant Fraction of Human Lens-Free Whole Eye and Liver.

The increasing incidence of ocular diseases has accelerated research into therapeutic interventions needed for the eye. Ocular enzymes play important roles in the metabolism of drugs and endobiotics. Various ocular drugs are designed as prodrugs that are activated by ocular enzymes. Moreover, ocular enzymes have been implicated in the bioactivation of drugs to their toxic metabolites. The key purpose of this study was to compare global proteomes of the pooled samples of the eye (n = 11) and the liver (n = 50) with a detailed analysis of the abundance of enzymes involved in the metabolism of xenobiotics and endobiotics. We used the postmitochondrial supernatant fraction (S9 fraction) of the lens-free whole eye homogenate as a model to allow accurate comparison with the liver S9 fraction. A total of 269 proteins (including 23 metabolic enzymes) were detected exclusively in the pooled eye S9 against 648 proteins in the liver S9 (including 174 metabolic enzymes), whereas 424 proteins (including 94 metabolic enzymes) were detected in both the organs. The major hepatic cytochrome P450 and UDP-glucuronosyltransferases enzymes were not detected, but aldehyde dehydrogenases and glutathione transferases were the predominant proteins in the eye. The comparative qualitative and quantitative proteomics data in the eye versus liver is expected to help in explaining differential metabolic and physiologic activities in the eye. SIGNIFICANCE STATEMENT: Information on the enzymes involved in xenobiotic and endobiotic metabolism in the human eye in relation to the liver is scarcely available. The study employed global proteomic analysis to compare the proteomes of the lens-free whole eye and the liver with a detailed analysis of the enzymes involved in xenobiotic and endobiotic metabolism. These data will help in better understanding of the ocular metabolism and activation of drugs and endobiotics.

Isolation of prolactin gene and its differential expression during metamorphosis involving eye migration of Japanese flounder Paralichthys olivaceus.

Eye migration during flatfish metamorphosis is driven by asymmetrical cell proliferation. To figure out Prolactin (PRL) function in this process, the full-length cDNA of prl was cloned from Japanese flounder (Paralichthys olivaceus) in our study. The deduced PRL protein shares highly conserved sequence with other teleosts, but has several amino acids loss compared with higher vertebrates, including amphibians, reptiles, avian and mammals. Spatio-temporal expression of prl gene displayed its extensive expression in the early development stages, while the limited expression of prl was observed in the pituitary, brain, and intestine of adult fish. In situ hybridization showed the asymmetrical distribution patterns of prl gene around the eyes during metamorphosis, which was coincident with the cell proliferation signals. Colchicine inhibited cell proliferation and reduced the prl gene expression, which indicates that PRL was involved in cell proliferation in the suborbital area of the migrating eye. The treatment of methimazole and 9-cis-retinoic acid respectively led to a reduction in the number of proliferating cells and the downregulation of prl expression, suggesting PRL was regulated by thyroid hormone signaling pathway and retinoic acid related signaling pathways. The results gave us a basic understanding of PRL function during flatfish metamorphosis.

Glucopyranoside flavonoids isolated from leaves of Spinacia oleracea (spinach) inhibit the formation of advanced glycation end products (AGEs) and aldose reductase activity (RLAR).

BACKGROUND AND PURPOSE: The formation and accumulation of advanced glycation end products (AGEs) and rat lens aldose reductase (RLAR) generated in the glycation process play an outstanding role in the complications of diabetes. Owing to the adverse effects of AGEs on diabetic patients, the search for new anti-AGE agents from plants without side effects has had significant interest from the researchers in the last decades for the development of a therapy that improves diabetic complications. Spinach could reverse the formation of AGEs and RLAR. This study aimed to investigate the ability of 10 known glucopyranosides flavonoids isolated from Spinacia oleracea on the formation of AGEs and RLAR in vitro and in vivo experiments. MATERIALS AND METHODS: Methanol extract of leaves of spinach was subjected to bioassay-guided fractionation using to silica gel column chromatographic followed by gel filtration by Sephadex LH-20. BSA glucose system and in vitro bioassays using rat lens aldose reductase (RLAR) were employed to evaluated inhibitory activity on the formation of AGEs. The induced diabetes in zebrafish by immersing in a 111 mM glucose solution for 14 days, revealed increased glycation of proteins in the eyes. Measurements of glycated hemoglobin and fructosamine were used to verify the anti-AGEs effect of the isolated flavonoids. KEY RESULTS: Through bioassay-guided fractionation of methanol extract of leaves spinach, ten known glucopyranoside flavonoids (1-10) have been isolated, and spectroscopic studies established their structures. Among the isolated compounds are: patuletin-3-O-(2"-coumaroylglucosyl)-(1→6)-[apiosyl-(1→2)]- ß-d-glucopyranoside (7), patuletin 3-O-(2"-feruloyl glucosyl)-(1→6)-[apiosyl-(1→2)]- ß-d-glucopyranoside (8), they have shown potent inhibition on AGEs formation, stronger than the positive controls used in the different experiments. CONCLUSION AND IMPLICATIONS: The findings indicated that glucopyranoside flavonoids found in Spinacia oleracea might have therapeutic potential for decreasing protein glycation, and might ameliorate AGE-related diabetic complications.

Ocular surface histopathological insult following sarin and VX exposure and potential treatments in the rat model.

Eye exposure to organophosphate (OP) chemical warfare irreversible acetylcholinesterase inhibitors, results in long-term miosis and impaired visual function. In contrast to the well-documented miotic and ciliary muscle spasm observed following chemical warfare, OP ocular exposure, little is known regarding the ocular surface histopathological insult. The aim of the present study was to determine the degree of the ocular surface insult following sarin or VX ocular exposure and to evaluate potential anti-cholinergic treatments in counteracting this insult. Rats that were whole body exposed to various sarin concentrations (0.049-43 µg/L; 5 min exposure), showed a dose-dependent miotic response and light reflex impairment. Following whole body sarin exposure, a dose dependent ocular surface histopathological insult was developed. A week following exposure to a low concentration of 0.05 µg/L, conjunctival pathology was observed, while corneal insult was noticed only following exposure to a concentration of 0.5 µg/L and above. Both tissues presented poorer outcomes when exposed to higher sarin concentrations. In contrast, eyes topically exposed to 1 µg sarin demonstrated no ocular insult a week following exposure. On the contrary, topical exposure to 1 µg VX resulted in a significant corneal insult. Anticholinergic treatments such as 0.1% atropine or 2% homatropine, given shortly following VX exposure, counteracted this insult. The results of this study show that not only do anti-cholinergic treatments counteract the miotic response, but also prevent the histopathological insult observed when given shortly following OP exposure.

An opsin 5-dopamine pathway mediates light-dependent vascular development in the eye.

During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.

Upregulation of calcitonin gene-related peptide, neuronal nitric oxide synthase, and phosphorylated extracellular signal-regulated kinase 1/2 in the trigeminal ganglion after bright light stimulation of the eye in rats.

Bright light stimulation of the eye activates trigeminal subnucleus caudalis (Vc) neurons in rats. Sensory information is conveyed to the Vc via the trigeminal ganglion (TG). Thus, it is likely that TG neurons respond to photic stimulation and are involved in photic hypersensitivity. However, the mechanisms underlying this process are unclear. Therefore, the hypothesis in this study is bright light stimulation enhances the excitability of TG neurons involved in photic hypersensitivity. Expressions of calcitonin gene-related peptide (CGRP) and neuronal nitric oxide synthase (nNOS) were significantly higher in TG neurons from 5 min to 12 h after photic stimulation of the eye. Phosphorylation of extracellular signal-regulated kinase1/2 (pERK1/2) was enhanced in TG neurons within 5 min after photic stimulation, while pERK1/2 immunoreactivity in satellite glial cells (SGCs) persisted for more than 12 h after the stimulus. Activation of SGCs was observed from 5 min to 2 h. Expression of CGRP, nNOS, and pERK1/2 was observed in small and medium TG neurons, and activation of SGCs and pERK1/2-immunoreactive SGCs encircling large TG neurons was accelerated after stimulation. These results suggest that upregulation of CGRP, nNOS, and pERK1/2 within the TG is involved in photic hypersensitivity.

Netarsudil ophthalmic solution 0.02% for the treatment of patients with open-angle glaucoma or ocular hypertension.

Once-daily (p.m.) netarsudil ophthalmic solution 0.02% (Rhopressa) is approved in the United States for lowering elevated intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension. Netarsudil, a Rho kinase (ROCK) inhibitor that lowers IOP primarily by increasing trabecular outflow, produces statistically and clinically significant reductions in mean IOP from baseline, with comparable effects on nocturnal and diurnal IOP. In three phase III trials of patients with elevated IOP, the ocular hypotensive efficacy of once-daily netarsudil 0.02% met the criteria for noninferiority to twice-daily timolol 0.5% at all time points over 3 months in patients with baseline IOP less than 25 mmHg. The most frequent adverse event (AE) was generally mild conjunctival hyperemia, the severity of which did not increase with continued dosing. Netarsudil was associated with minimal treatment-related serious or systemic AEs, likely due to the lack of systemic exposure. This report summarizes the available preclinical and clinical data on netarsudil.

Esterase activity in porcine and albino rabbit ocular tissues.

Corneal esterases are utilized in the activation of topically applied ester prodrugs. Esterases may also be involved in the metabolism of drugs in posterior eye tissues, but their physiological activity is unknown. Furthermore, extrapolation of the esterase activity from protein level to the tissues is missing. The aims of the current study were to determine esterase activities in porcine and albino rabbit ocular tissues, calculate the activities for whole tissues and compare esterase activity between the species. We conducted a hydrolysis study with ocular tissue homogenates using an esterase probe substrate 4-nitrophenyl acetate. The hydrolysis rates were first normalized to protein content and then scaled to whole tissues. The hydrolytic rate normalized to protein content was high in the cornea and iris-ciliary body and low in the lens and aqueous humor, and in general, the rabbit tissues had higher hydrolytic rates than the porcine ones. Esterase activity scaled to whole tissue was high in cornea and iris-ciliary body and low in aqueous humor and retinal pigment epithelium in both species. The current study revealed differences in esterase activities among the ocular tissues and the species. This basic knowledge on ocular esterases provides background information particularly for posterior segment drug development.

Nitric oxide-donating compounds for IOP lowering in glaucoma. / Donadores de óxido nítrico como hipotensores en glaucoma.

INTRODUCTION: An elevated intraocular pressure (IOP) remains the main risk factor for progression of glaucoma upon which we can efficiently act. Pharmacological strategies to reduce IOP are directed towards the reduction of aqueous humour (AH) production and/or the increase in AH drainage through the uveoscleral pathway. However, there are no drugs currently available as first-line treatment to increase AH outflow primarily via the conventional route. Ocular nitric oxide (NO) production takes place in AH outflow pathways and in the ciliary muscle, modulating the cellular response to elevated IOP. METHODS: This review describes the mechanism of action of endogenous NO and NO-donating compounds that are under research. It includes information regarding pre-clinical and clinical studies previously conducted with these compounds, discussing their role and therapeutic potential in the pharmacological treatment of ocular hypertension in glaucoma. RESULTS: The topical ocular administration of NO-donating compounds significantly lowered IOP and maintained it in animal models of glaucoma and subjects with ocular hypertension. CONCLUSIONS: The mechanism of action of these compounds is novel and scientific evidence that shows promising results. However, there is a need for more comprehensive studies to assess long-term safety and tolerability in order to properly evaluate their use in chronic therapies.

Cyclooxygenase-2 expression in the eyes of cats with and without uveitis.

OBJECTIVE To characterize the distribution and intensity of cyclooxygenase (COX)-2 expression in the eyes of cats with and without uveitis and to determine whether COX-2 expression is correlated with severity of inflammation. SAMPLES Archived ocular tissue specimens from 51 cats with and 10 cats without ocular disease. PROCEDURES Specimens from only 1 eye were evaluated for each cat. Specimens were stained with H&E stain or immunohistochemical stain for detection of COX-2 and reviewed. For each eye, the type, severity, and distribution of inflammation and the distribution and intensity of COX-2 expression were determined for the uvea and other ocular tissues. Correlation between COX-2 expression and inflammation severity was also assessed. RESULTS COX-2 was not expressed in any nondiseased eye. Of the 51 diseased eyes, 20 had histologic evidence of lymphocytic-plasmacytic uveitis, 13 had neutrophilic uveitis, 11 had diffuse iris melanoma with uveitis, and 7 had diffuse iris melanoma without uveitis. Of the 44 eyes with uveitis, COX-2 was detected in the uvea of 16, including 11 eyes with lymphocytic-plasmacytic uveitis, 4 with neutrophilic uveitis, and 1 with diffuse iris melanoma-induced uveitis. Inflammation was severe, moderate, or mild in 10, 5, and 1 of those eyes, respectively. Cyclooxygenase-2 was detected in the cornea of 21 eyes with uveitis and 1 eye with diffuse iris melanoma without uveitis. Uveitis severity was positively correlated with COX-2 expression in both the uvea and cornea. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that COX-2 is an inflammatory mediator in feline uveitis but not diffuse iris melanoma.

Differential Expression of Seven De-sumoylation Enzymes (SENPs) in Major Ocular Tissues of Mouse Eye.

PURPOSE: Protein Sumoylation is one of the most important and prevalent posttranscriptional modification. Increasing evidence have shown that the SENPs (sentrin/SUMOspecific proteases) are critical for steady-state levels of SUMO modification of target proteins, and protein de-sumoylation modulates a great diversity of biological processes including transcription, development, differentiation, neuroprotection, as well as pathogenesis. In the vertebrate eye, we and others have previously shown that sumoylation participated in the differentiation of major ocular tissues including retina and lens. However, the biological significance of seven SENP enzymes: SENP1 to 3 and SENP5 to 8 have not be fully investigated in the ocular tissues. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: At the mRNA level, all SENPs were highly expressed in retina, and much reduced expression patterns in cornea, lens epithelium and lens fiber. At the protein level, SENP1 to -3, and SENP6 were highly abundant in cornea, while SENP5, SENP7 and SENP8 were enriched in retina, and these SENPs were relatively less abundant in lens tissues. CONCLUSION: Our results for the first time established the differentiation expression patterns of the 7 de-sumoylation enzymes (SENPs), which provides a basis for further investigation of protein desumoylation functions in vertebrate eye.

Localization Analysis of Seven De-sumoylation Enzymes (SENPs) in Ocular Cell Lines.

PURPOSE: It is now well established that protein sumoylation acts as an important regulatory mechanism modulating functions over three thousand proteins. In the vision system, protein conjugation with SUMO peptides can regulate differentiation of multiple ocular tissues. Such regulation is often explored through analysis of biochemical and physiological changes with various cell lines in vitro. We have recently analyzed the expression levels of both mRNAs and proteins for seven de-sumoylation enzymes (SENPs) in five major ocular cell lines. In continuing the previous study, here we have determined their cellular localization of the seven de-sumoylation enzymes (SENP1, 2, 3, 5, 6, 7 and 8) in the above 5 major ocular cell lines using immunocytochemistry. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The localization of the 7 major de-sumoylation enzymes (SENPs) in the 5 major ocular cell lines were determined with immunohistochemistry. The images were captured with a Zeiss LSM 880 confocal microscope. RESULTS: 1) The SENP1 was localized in both cytoplasm and nucleus of 3 human ocular cell lines, FHL124, HLE and ARPE-19; In N/N1003A and αTN4-1, SENP 1 was more concentrated in the cytoplasm. SENP1 appears in patches; 2) SENP2 was distributed in both cytoplasm and nucleus of all ocular cell lines in patches. In HLE and ARPE-19 cells, SENP2 level was higher in nucleus than in cytoplasm; 3) SENP3 was almost exclusively concentrated in the nuclei in all ocular cells except for N/N1003A cells. In the later cells, a substantial amount of SENP3 was also detected in the cytoplasm although nuclear SENP3 level was higher than the cytoplasmic SENP3 level. SENP3 appeared in obvious patches in the nuclei; 4) SENP5 was dominantly localized in the cytoplasm (cellular organelles) near nuclear membrane or cytoplasmic membrane ; 5) SENP6 was largely concentrated in the nuclei of all cell lines except for αTN4-1 cells. In the later cells, a substantial amount of SENP6 was also detected in the cytoplasm although nuclear SENP6 level was higher than the cytoplasmic SENP6 level. 6) SENP7 has an opposite localization pattern between human and animal cell lines. In human cell lines, a majority of SENP7 was localized in nuclei whereas in mouse and rabbit lens epithelial cells, most SENP7 was distributed in the cytoplasm. SENP8 was found present in human cell lines. The 3 human ocular cell lines had relatively similar distribution pattern. In FHL124 and ARPE-19 cells, SENP8 was detected only in the cytoplasm, but in HLE cells, patches of SENP8 in small amount was also detected in the nuclei. CONCLUSIONS: Our results for the first time defined the differential distribution patterns of seven desumoylation enzymes (SENPs) in 5 major ocular cell lines. These results help to understand the different functions of various SENPs in maintaining the homeostasis of protein sumoylation patterns during their functioning processes.

Analysis of the Differential Expression Patterns of Sumoylation Enzymes E1, E2 and E3 in Ocular Cell Lines.

PURPOSE: Protein sumoylation is a well established regulatory mechanism to control many cellular processes such as chromatin structure dynamics, transcriptional regulation of gene expression, cell proliferation and differentiation, cell transformation and carcinogenesis, autophagy and senescence. In the vertebrate vision system, we and others have revealed that sumoylation plays important roles in regulating differentiation of several ocular tissues during eye development. To further elucidate the functional mechanisms of sumoylation, in vitro assay systems are needed. Currently, the five major cell lines including αTN4-1, FHL124, HLE, N/N1003A and ARPE-19 have been extensively used to test the biochemical and molecular aspects of normal vision physiology and various disease processes. Thus, we conducted the study on the expression patterns of the three types of sumoylation enzymes, the activating enzymes SAE1 and UBA2, the conjugating enzyme UBC9, and the ligating enzymes such as RanBP2 and PIAS1 in these ocular cell lines. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: we have obtained the following results: 1) For the mRNAs encoding E1 SAE1 and UBA2, E2 UBC9 and E3 PIAS1, the highest level of expression was observed in αTN4-1 cells; For the mRNA encoding RanBP2, the highest level of expression was detected in N/N1003A cells; 2) In contrast to the mRNA expression patterns, a similar level of the SAE1 protein was observed in the all five cell lines, and so is true with UBA2 protein in all cells except for N/N1003A where over fourfold of enrichment in UBA2 protein was observed compared with other cell lines; 3) A similar level of UBC9 protein was also detected in all cells except for N/N1003A where more than one-fold of decrease in UBC9 level was found compared with other cell lines; 4) For E3 ligases, we did not identify the regular PIAS1 band in N/N1003A cells, the remaining cells have a level of PIAS1 with difference of less than 0.6-fold; all cells except for FHL124 cells have a similar level of RanBP2, and a 70% drop in RanBP2 was observed in FHL124 cell. CONCLUSIONS: Our determination of the differential expression patterns of the three types of sumoylation enzymes in the 5 ocular cell lines help to understand sumoylation functions in vertebrate eye.

Localization Patterns of Sumoylation Enzymes E1, E2 and E3 in Ocular Cell Lines Predict Their Functional Importance.

PURPOSE: It is well established now that protein sumoylation acts as an important regulatory mechanism mediating control of ocular development through regulation of multiple transcription factors. Yet the functional mechanisms of each factor modulated remain to be further explored using the available in vitro systems. In this regard, various ocular cell lines including HLE, FHL124, αTN4-1, N/N1003A and ARPE-19 have been demonstrated to be useful for biochemical and molecular analyses of normal physiology and pathogenesis. We have recently examined that these cell lines express a full set of sumoylation enzymes E1, E2 and E3. Following this study, here we have examined the localization of these enzymes and determined their differential localization patterns in these major ocular cell lines. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The localization of the 3 major sumoylation enzymes in the 5 major ocular cell lines were determined with immunohistochemistry. The images were captured with a Zeiss LSM 880 confocal microscope. RESULTS: we have obtained the following results: 1) The sumoylation enzymes SAE1, UBC9 and PIAS1 are distributed in both nucleus and cytoplasm, with a much higher level concentrated in the nucleus and the neighboring cellular organelle zone in all cell lines; 2) The sumoylation enzyme UBA2 was highly concentrated in both cytoplasm membrane, cytoskeleton and nucleus of all cell lines; 3) The ligase E3, RanBP2 was exclusively localized in the nucleus with homogeneous distribution. CONCLUSIONS: Our results for the first time established the differential localization patterns of the three types of sumoylation enzymes in 5 major ocular cell lines. Our establishment of the differential localization patterns of the three types of sumoylation enzymes in these cell lines help to predict their functional importance of sumoylation in the vision system. Together, our results demonstrate that these cell lines can be used for assay systems to explore the functional mechanisms of sumoylation mediating ocular development and pathogenesis.

Short-term and persistent impacts on behaviors related to locomotion, anxiety, and startle responses of Japanese medaka (Oryzias latipes) induced by acute, sublethal exposure to chlorpyrifos.

Although most exposures to chlorpyrifos (CPF) in natural flowing waters are brief and episodic, there have been a few reports of the persistence of abnormal fish behaviors caused by such acute exposure. The present study focused on the behavioral and biochemical responses of Japanese medaka (Oryzias latipes) to acute, sublethal exposure to CPF, as well as the persistence of the effects during a 3-week recovery test in CPF-free water. The medaka became hyperactive and exhibited an elevated anxiety state after a 4-day exposure to 0.024mg/L of CPF, but they recovered from these abnormal behavioral responses within 7days of recovery treatment. In contrast, persistent impacts on some startle responses to a sudden stimulation (induced by a ball drop) were observed in medaka exposed to CPF. The reaction latency did not change immediately after the 4-day exposure, but was significantly prolonged by as much as 21days after the termination of exposure. The post-stimulus swimming distance within 5s significantly decreased on the day immediately after the 4-day exposure, but it significantly increased after 7days of recovery treatment. The activity of acetylcholinesterase (AChE) in the brains of medaka was significantly inhibited on the day immediately after the 4-day exposure, but it returned to 80% and 110% of that in control fish on days 7 and 21 of the recovery period, respectively. However, AChE activities in the eyes of exposed medaka were persistently inhibited and declined to 33%, 71%, and 72% of that in control fish on days 0 (immediately after the 4-day exposure), 7, and 21 of recovery, respectively. Correlation analysis suggested that the changes of AChE activities in the brains of medaka may underlie some of the observed acute behavioral changes, and the changes of AChE activities in the eyes may contribute to the persistence of the abnormalities in the reaction latency of the startle response. Our findings suggest that medaka need a long time to recover from acute, sublethal exposure to CPF, and the persistence of the behavioral abnormalities might affect their fitness in natural habitats.

Investigation of Ocular Bioactivation Potential and the Role of Cytochrome P450 2D Enzymes in Rat.

BACKGROUND: Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat open-angle glaucoma. Ocular administration of timolol in low doses (0.5% w/v in the form of eye drops) has led to challenges for in vivo metabolite identification. An understanding of drug metabolism in the eye is important for clinical ocular therapeutics and potential drug candidates. METHODS: We aimed to investigate the metabolism of timolol in rat ocular and liver S9 fractions, as well as rat ocular tissue and plasma following a 0.5% topical (ocular) dose of timolol. We explored the potential in vitro metabolic bioactivation in the eye/liver by conducting trapping studies for putative aldehyde and iminium ion intermediates that may arise from the morpholine functionality. RESULTS: Oxidative metabolism of timolol to its major metabolite (M4) in ocular S9 and recombinant rat cytochrome P450 (CYP) isoforms supports the possible role of rat ocular CYP2D2, 2D4, and/or 2D18. Observation of N-acetyl-timolol (M5) is suggestive that the ocular N-acetyltransferases may also play a larger role in ocular disposition of timolol, a previously unreported finding. This research is the first comprehensive report of in vitro ocular metabolism of timolol in rat. CONCLUSION: This study also indicates that in vitro hepatic metabolism is over-predictive of ocular metabolism following topically ocular dosed timolol. The research, herein, highlights the eye as an organ capable of first pass metabolism for topical drugs. Thus, new ophthalmologic considerations for studying and designing long term topical therapies in preclinical species are needed in drug discovery.

MAPK signaling pathways in eye wounds: Multifunction and cooperation.

As is widely distributed in eukaryotic cells, the mitogen-activated protein kinase (MAPK) signaling pathway family plays an inevitable role in diverse cellular processes, being capable of responding to particular physiological reactions induced by multiple extracellular signals or stimuli, such as protean concentrations, ischemia/reperfusion, and inflammation. The physiological reactions mediated by the MAPK signaling pathway contribute to the progression and healing of eye wounds. Meanwhile, several pathways in the MAPK family can cooperate with each other and establish distinct responses to different, or even the same, stimuli and, thus, more attention may be paid to the pathway in future research.

Ocular non-P450 oxidative, reductive, hydrolytic, and conjugative drug metabolizing enzymes.

Metabolism in the eye for any species, laboratory animals or human, is gaining rapid interest as pharmaceutical scientists aim to treat a wide range of so-called incurable ocular diseases. Over a period of decades, reports of metabolic activity toward various drugs and biochemical markers have emerged in select ocular tissues of animals and humans. Ocular cytochrome P450 (P450) enzymes and transporters have been recently reviewed. However, there is a dearth of collated information on non-P450 drug metabolizing enzymes in eyes of various preclinical species and humans in health and disease. In an effort to complement ocular P450s and transporters, which have been well reviewed in the literature, this review is aimed at presenting collective information on non-P450 oxidative, hydrolytic, and conjugative ocular drug metabolizing enzymes. Herein, we also present a list of xenobiotics or drugs that have been reported to be metabolized in the eye.

APC/CFzr/Cdh1-Dependent Regulation of Planar Cell Polarity Establishment via Nek2 Kinase Acting on Dishevelled.

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase, well known for its role in cell-cycle progression. However, it has been linked to additional functions, mainly in neuronal contexts, when using the co-activator Cdh1/Fzr. Here, our data indicate a post-mitotic requirement for the APC/CFzr/Cdh1 in epithelial cell patterning and planar cell polarity (PCP) in Drosophila. PCP signaling is critical for development by establishing cellular asymmetries and orientation within the plane of an epithelium, via differential localization of distinct complexes of core PCP factors. Loss of APC/C function leads to reduced levels of Dishevelled (Dsh), a core PCP factor. The effect of APC/C on Dsh is mediated by Nek2 kinase, which can phosphorylate Dsh and is a direct APC/CFzr/Cdh1 substrate. We have thus uncovered a pathway of regulation whereby APC/CFzr/Cdh1 negatively regulates Nek2, which negatively regulates Dsh, to ensure its proper stoichiometric requirement and localization during PCP establishment.