Characteristics of corneal phospholipidosis induced by topical ocular application of chloroquine and amiodarone in rabbits.

Several cationic-amphiphilic drugs such as chloroquine and amiodarone are known to induce phospholipidosis in the cornea by systemic administration. However, the characteristics of ophthalmological and pathological changes when phospholipidosis-inducing drugs are topically applied have not been well studied. This study was conducted to investigate the characteristics of corneal changes caused by topical application of chloroquine and amiodarone to Japanese white rabbits. The changes were evaluated by ophthalmological, histopathological, and ultrastructural examinations. An in vivo confocal microscopy was also applied to the chloroquine-treated corneas. In both chloroquine- and amiodarone-treated corneas, diffuse cloudiness was observed by slit-lamp biomicroscopy, and its transparency increased with duration of dosing. Confocal microscopy showed punctate dots in the corneal epithelium. Histopathologically, cytoplasmic vacuolation was found in the corneal epithelium and keratocytes in both chloroquine- and amiodarone-treated eyes. Furthermore, foamy cytoplasm of the corneal endothelium was observed in the chloroquine-treated eyes. Ultrastructural examination showed multi-lamellar inclusion bodies or membrane-like debris in the lysosome-like vacuoles in the cytoplasm of corneal cells, which is a characteristic of the lesions of phospholipidosis. These changes disappeared after a withdrawal period. Continuous dosing of chloroquine resulted in corneal erosion and focal corneal opacity as shown by gross observation and slit-lamp biomicroscopy. Confocal microscopy could detect the corneal changes prior to the appearance of these ophthalmological changes. The present study showed that phospholipidosis caused by ocular administration of chloroquine and amiodarone first induces reversible diffuse corneal cloudiness. Confocal microscopy is a useful method for monitoring induction of corneal phospholipidosis.

The putative promoters of germ cell-specific genes and Nanog are hypomethylated in chicken sperm.

Germ cell-specific genes such as Ddx4, Dnd1, and Dazl play critical roles in the proliferation and survival of germ cells. However, the methylation state of the promoter in mature germ cells is still unknown. Here, we investigated the methylation levels of these genes and the pluripotency marker gene Nanog in chicken sperm as compared with the Alb gene in the liver. CpG islands and/or promoter motifs such as TATA box, GC box and CAAT box were found within the putative promoter regions that we identified. By using the bisulfite reaction, CpG sites in the putative promoters were converted, and they were analyzed by sequencing. The putative promoters of Ddx4, Dnd1, Dazl and Nanog showed very low methylation levels in sperm, but they were highly methylated in the liver. Conversely, the Alb gene promoter was highly methylated in sperm and hypomethylated in the liver. However, no transcripts of Ddx4, Dnd1, Dazl and Nanog were detected in sperm or the liver. Also, no transcripts of Dnmt1 and Dnmt3a were detected in sperm. Our present results may indicate that these germ cell-specific genes and the pluripotency marker gene are ready to express any time after fertilization. Our findings showing that low methylation and selective DNA methylation of specific genes are present in chicken sperm contribute to our understanding of fertilization and embryogenesis of birds.

Efficacy and safety of topical azithromycin therapy in patients with blepharitis and meibomian gland dysfunction.

PURPOSE: To assess the effects of 1% azithromycin ophthalmic solution (AZM) in patients with bacterial blepharitis accompanied by meibomian gland dysfunction (MGD). STUDY DESIGN: A multicenter, single arm, prospective interventional study. METHODS: AZM was administered to the affected eyes twice daily for the first 2 days and once daily for the subsequent 12 days. Lid margin hyperaemia/redness, collarette at the root of the eyelashes, conjunctival hyperaemia, foreign body sensation, and epiphora were assessed on Days 1, 14, and 28. The Dry Eye-related Quality of Life Score (DEQS) and objectives related to MGD, including lid vascularity, lid margin irregularity, foaming, lid plugging, keratoconjunctival disorders, Marx line, meibum grade, and tear breakup time, were also assessed. Bacterial culture of the conjunctival sac and meibum was performed on Days 1 and 14. RESULTS: Twenty-four eyes of 24 patients (10 men/14 women, mean age 72.3 ± 13.2) were included. On Days 14 and 28, the total score, lid vascularity, lid plugging, and meibum grade showed significant improvement (p < 0.05). On Day 1, 71 strains were isolated from 22 of the 24 eyes (91.7%). Cutibacterium acnes, Corynebacterium spp., and Staphylococci were detected at high frequencies. The overall disappearance rates of the bacteria in the conjunctival sac and meibum at the end of treatment were 65.7% and 58.3%, respectively. No serious ocular or systemic adverse events were observed. CONCLUSION: Fourteen-day treatment with AZM was effective in patients with blepharitis accompanied by MGD, and the efficacy of AZM persisted for a period after the treatment.

FSH induces the development of circadian clockwork in rat granulosa cells via a gap junction protein Cx43-dependent pathway.

The present study was designed to assess the relationship between gap junctions and the maturation of a clock system in rat granulosa cells stimulated by follicle-stimulating hormone (FSH). Immature and mature granulosa cells were prepared by puncturing the ovaries of diethylstilbestrol- and equine chorionic gonadotropin (eCG)-treated mouse Period2 (Per2)-dLuc reporter gene transgenic rats, respectively. Mature granulosa cells exposed to dexamethasone (DXM) synchronization displayed several Per2-dLuc oscillations and a rhythmic expression of clock genes. Intriguingly, we observed clear evidence that the FSH stimulation significantly increased the amplitude of Per2 oscillations in the granulosa cells, which was confirmed by the elevation of the Per2 and Rev-erbα (Nr1d1) mRNA levels. FSH also induced a major phase-advance shift of Per2 oscillations. The mature granulosa cells cultured for 2 days with FSH expressed higher mRNA levels of Per2, Rev-erbα, Bmal1 (Arnt1), Lhcgr, and connexin (Cx) 43 (Gja1) compared with the immature granulosa cells. Consistently, our immunofluorescence results revealed abundant Cx43 protein in antral follicles stimulated with eCG and weak or no fluorescence signal of Cx43 in primary and preantral follicles. Similar results were confirmed by Western blotting analysis. Two gap junction blockers, lindane and carbenoxolone (CBX), significantly decreased the amplitude of Per2 oscillations, which further adhered significant decreases in Per2 and Rev-erbα transcript levels. In addition, both lindane and CBX induced a clear phase-delay shift of Per2 oscillations. These findings suggest that FSH induces the development of the clock system by increasing the expression of Cx43.

A reaction mechanism-based prediction of mutagenicity: α-halo carbonyl compounds adduct with DNA by SN2 reaction.

Most of the α-halo carbonyl (AHC) compounds tend to be predicted as mutagenic by structure-activity relationship based on structural category only, because they have an alkyl halide structure as a structural alert of mutagenicity. However, some AHC compounds are not mutagenic. We hypothesized that AHC reacts with DNA by SN2 reaction, and the reactivity relates to mutagenicity. As an index of SN2 reactivity, we focused on molecular orbitals (MOs), as the direction and position of two molecules in collision are important in the SN2 reaction. The MOs suitable for SN2 reaction (SN2MOs) were selected by chemical-visual inspection based on the shape of the MO. We used the level gap and the energy gap between SN2MO and the lowest unoccupied molecular orbital as the descriptors of SN2 reactivity. As the results, SN2 reactivity related to mutagenicity and we were able to predict mutagenicity of 20 AHC compounds with 95.0% concordance. It was suggested that SN2 reaction is a reaction mechanism of AHC compounds and DNA in the mutagenic process. The method allows for discrimination among structurally similar compounds by combination with quantitative structure-activity relationships. The combination approach is expected to be useful for the mutagenic assessment of pharmaceutical impurities.

Temporal and spatial differential expression of chicken germline-specific proteins cDAZL, CDH and CVH during gametogenesis.

The Deleted in Azoospermia-Like (DAZL) protein coded by Dazl gene is a germline-specific RNA-binding protein essential for gametogenesis in vertebrates, and the chicken Dazl gene has also been identified in primordial germ cells (PGCs). However, the temporal and spatial expression of chicken DAZL (cDAZL) and its molecular role in germ cell development remain enigmatic. Here, we investigated the subcellular distribution and expression of cDAZL at the various stages by using a polyclonal antibody raised against its C-terminal region and compared them with those of additional germline-specific proteins chicken vasa homologue (CVH) and chicken dead end homologue (CDH). Western blot analysis for cDAZL revealed a single band in the embryonic gonads and premature chicken testis, whereas no band was detected in the premature chicken ovary. Fluorescent immunohistochemistry revealed that cDAZL was present in the nucleus and cytoplasm of circulating PGCs. Cells positive for cDAZL and CVH coexisted in the embryonic gonads and premature chicken testis, in which they were distributed near the basement membrane of seminiferous tubules. Of interest, cDAZL was not found in the premature chicken ovary, whereas CVH and CDH were present in germ cells. Collectively, three germline-specific proteins are expressed in chicken germ cells, but their patterns of expression are temporally and spatially distinct.