Downregulation of fascin induces collective cell migration in triple­negative breast cancer.

Breast cancer (BC) is one of the most common types of cancer affecting female patients. Triple­negative BC (TNBC) is an aggressive subtype. Fascin, an actin­bundling protein, serves a significant role in cancer metastasis. Fascin overexpression is associated with poor prognosis of BC. To confirm the relationship between fascin expression and BC malignancy, the present study reviewed clinical data from 100 Japanese patients with BC and performed fresh immunohistochemical fascin examination of tissue samples. Statistical analyses showed metastasis or recurrence in 11 of 100 patients and a significant association between high fascin expression and poor prognosis. The TNBC subtype was also associated with high fascin expression. However, a few cases developed poor prognosis regardless of negative or slightly positive fascin expression. The present study established fascin knockdown (FKD) MDA­MB­231, a TNBC cell line, and investigated morphological effects of fascin on TNBC cells. FKD cells exhibited cell­cell connections and bulbous nodules of various sizes on the cell surface. Conversely, non­FKD MDA­MB­231 cells exhibited loose cell­cell connections with numerous filopodia on the cell surface. Filopodia, actin­rich plasma membrane protrusions, are composed of fascin and control cell­cell interaction, migration and wound healing. Cancer metastasis is conventionally classified into two mechanisms: single and collective cell migration. Fascin increases cancer metastasis by single cell migration via filopodia on the cell surface. However, the present study suggested that following FKD, TNBC cells lost filopodia and exhibited collective cell migration.

Evaluation of Clinical and Immunohistochemical Factors Relating to Melanoma Metastasis: Potential Roles of Nestin and Fascin in Melanoma.

For melanoma treatment, an early diagnosis and a complete resection of the primary tumor is essential. In addition, detection of factors that may be related to metastasis is indispensable. A total of 30 Japanese patients with Stage I or II melanoma, diagnosed according to the classification of the American Joint Committee on Cancer, are included in this study. Clinical background (sex, onset age, primary tumor area, existence of remaining cancer cells at the resected tissue margin, and treatment after the primary surgery) and immunohistochemical staining (Nestin and Fascin) on the resected tissue were examined to detect factors statistically related to metastasis. The analysis result has shown that older onset age and positive immunohistochemical expressions of Nestin and Fascin are statistically related to metastasis. To facilitate meticulous observation of Nestin and Fascin expression at different timing (e.g., onset and metastasis), double immunofluorescence staining was performed. Nestin is a class VI intermediate filament protein, initially detected in neural stem cells. Fascin is an actin-bundling protein which regulates cell adhesion, migration and invasion. Nestin and Fascin are suggested to relate to melanoma metastasis, however, the potential role of Fascin is controversial. Analysis of variations in Fascin expression detected in this study may contribute to further investigations concerning potential roles of Fascin for progression of melanoma. This is the first study to report double immunofluorescent staining of Nestin and Fascin in melanoma. Nestin and Fascin double-positive melanoma cells were detected.

Fascin-1 is associated with recurrence in solitary fibrous tumor/hemangiopericytoma.

Fascin-1, an actin-bundling protein, is associated with poor prognosis in patients with various types of human carcinoma. However, research is limited on the role of fascin-1 in sarcoma. Solitary fibrous tumor (SFT) and hemangiopericytoma (HPC) are rare sarcomas derived from the mesenchyme. Although the prognosis of SFT/HPC is generally favorable, fatalities are possible with repeated recurrence and distant metastasis. The current study included a total of 20 Japanese patients, who were diagnosed with SFT/HPC and underwent surgery at Kochi University Hospital from January 2000 to December 2019. The statistical relationship between recurrence and the following variables were examined: Sex, age of onset, tumor origin, tumor size, necrosis, mitosis ≥1/10 high power field (HPF; magnification, x400), Ki-67 >5% and Fascin-1. A significant association was determined between recurrence and necrosis, mitosis ≥1/10 HPF (magnification, x400), Ki-67 >5%, and Fascin-1 ≥'strongly positive' (P<0.05). The results demonstrated that Fascin-1 immunostaining may be a highly effective and useful evaluation factor for predicting poor prognosis in patients with SFT/HPC, a fatal sarcoma of humans.

Absence of histopathological changes in the retina of zebrafish treated with sodium iodate.

In ophthalmological research, the use of zebrafish to investigate visual behaviors has been increasing, but can produce misleading, false-positive results if compounds adversely affect their motor functions or central nervous system. Therefore, histological analysis to identify a target organ is important in zebrafish toxicity assay. We investigated the retinal degeneration in zebrafish, using typical retinal toxicants, mainly sodium iodate and N-methyl-N-nitrosourea (MNU). No histopathological changes were found after sodium iodate exposure at 1.0 mM for 5 or 7 days in the retina of larval, juvenile, and adult zebrafish. There were also no obvious histopathological changes in the retina of adult zebrafish at 0.1 mM, even after 30 days treatment with sodium iodate. In addition, many proliferating cell nuclear antigen-positive cells were found not only in the ciliary marginal zone, but also in the outer nuclear layer, especially in larval and juvenile zebrafish with or without sodium iodate exposure. However, the concentrations of iodine in the blood and the eyeballs of adult zebrafish increased remarkably after the treatment. General retinal damage emerged after MNU exposure at 150 mg/l for 60 min in adult zebrafish, but first pyknotic cells appeared in the inner nuclear layer and the ganglion cell layer. Our findings indicate that zebrafish retina have a different reactivity pattern from mammalian animals against some retinal toxicants, and in them it is difficult to detect histopathological changes.

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.

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.

Establishment of an in silico phospholipidosis prediction method using descriptors related to molecular interactions causing phospholipid-compound complex formation.

Although phospholipidosis (PLD) often affects drug development, there is no convenient in vitro or in vivo test system for PLD detection. In this study, we developed an in silico PLD prediction method based on the PLD-inducing mechanism. We focused on phospholipid (PL)-compound complex formation, which inhibits PL degradation by phospholipase. Thus, we used some molecular interactions, such as electrostatic interactions, hydrophobic interactions, and intermolecular forces, between PL and compounds as descriptors. First, we performed descriptor screening for intermolecular force and then developed a new in silico PLD prediction using descriptors related to molecular interactions. Based on the screening, we identified molecular refraction (MR) as a descriptor of intermolecular force. It is known that ClogP and most-basic pKa can be used for PLD prediction. Thereby, we developed an in silico prediction method using ClogP, most-basic pKa, and MR, which were related to hydrophobic interactions, electrostatic interactions, and intermolecular forces. In addition, a resampling method was used to determine the cut-off values for each descriptor. We obtained good results for 77 compounds as follows: sensitivity = 95.8%, specificity = 75.9%, and concordance = 88.3%. Although there is a concern regarding false-negative compounds for pKa calculations, this predictive ability will be adequate for PLD screening. In conclusion, the mechanism-based in silico PLD prediction method provided good prediction ability, and this method will be useful for evaluating the potential of drugs to cause PLD, particularly in the early stage of drug development, because this method only requires knowledge of the chemical structure.

Establishment of an in silico phototoxicity prediction method by combining descriptors related to photo-absorption and photo-reaction.

One of the mechanisms of phototoxicity is photo-reaction, such as reactive oxygen species (ROS) generation following photo-absorption. We focused on ROS generation and photo-absorption as key-steps, because these key-steps are able to be described by photochemical properties, and these properties are dependent on chemical structure. Photo-reactivity of a compound is described by HOMO-LUMO Gap (HLG), generally. Herein, we showed that HLG can be used as a descriptor of the generation of reactive oxygen species. Moreover, the maximum-conjugated π electron number (PENMC), which we found as a descriptor of photo-absorption, could also predict in vitro phototoxicity. Each descriptor could predict in vitro phototoxicity with 70.0% concordance, but there was un-predicted area found (gray zone). Interestingly, some compounds in each gray zone were not common, indicating that the combination of two descriptors could improve prediction potential. We reset the cut-off lines to define positive zone, negative zone and gray zone for each descriptor. Thereby we overlapped HLG and PENMC in a graph, and divided the total area to nine zones with cut-off lines of each descriptor. The rules to prediction were decided to achieve the best concordance, and the concordances were improved up to 82.8% for self-validation, 81.6% for cross-validation. We found common properties among false positive or negative compounds, photo-reactive structure and photo-allergenic, respectively. In addition, our method could be adapted to compounds rich in structural diversity using only chemical structure without any statistical analysis and complicated calculation.

Pharmacokinetic features of difluprednate ophthalmic emulsion in rabbits as determined by glucocorticoid receptor-binding bioassay.

PURPOSE: Difluprednate (6α,9-difluoro-11ß,17,21-trihydroxy-1,4-pregnadiene-3,20-dione 21-acetate 17-butyrate, DFBA) has long been used as an anti-inflammatory dermatological agent. The main objectives of the current study were to evaluate the pharmacokinetic and pharmacodynamic features of DFBA when used as an ophthalmic agent, and to compare these features with those of other common ophthalmic agents, to determine which has the highest activity. METHODS: A glucocorticoid (GC) receptor-binding test was performed to evaluate GC receptor-binding activity (GCRBA, the index of pharmacological effect). Using this information, we calculated dose-response curves, IC(50) values, and K(d) values to evaluate each drug's K(i) value. Finally, we performed studies in live rabbits to compare the activity of 4 formulations [0.002%, 0.01%, or 0.05% DFBA, or an ophthalmic solution of 0.1% betamethasone sodium phosphate (BMP)] at 4 time points (0.5, 1, 2, 4 h). At each time point, blood and eye samples were taken so that C(max) (the maximum equivalent concentration of the active DFBA metabolite, DFB), T(max) (the time at which C(max) was measured), and the area under the concentration-time curve could be compared across the 4 formulations. RESULTS: BMP had the highest K(i) value (8.4 × 10(-8) nmol/L), whereas DFB had the lowest (6.1 × 10(-11) nmol/L). The GCRBA of DFBA was intermediate to these 2 values (7.8 × 10(-10) nmol/L). Instillation of the DFBA ophthalmic emulsion in the eyes of rabbits led to dose-dependent increases in GCRBA, which was mostly attributable to the activity of DFB. The 0.05% DFBA ophthalmic emulsion elicited the greatest response in both aqueous humor and iris/ciliary body tissues, though there were no significant dose-dependent differences in GCRBA in plasma samples. CONCLUSIONS: The 0.05% DFBA ophthalmic emulsion appears to be an effective and safe anti-inflammatory treatment in ocular tissues. It is comparable, and possibly even superior, to the 0.1% BMP solution, and may be particularly useful in cases of severe disease where treatment with BMP solution alone is insufficient.

Ocular distribution of difluprednate ophthalmic emulsion 0.05% in rabbits.

PURPOSE: To evaluate ocular distribution and excretion of tritium-labeled difluprednate ((3)H-DFBA) ophthalmic emulsion 0.05% after a single or repeated instillation to pigmented rabbit eyes. METHODS: (3)H-DFBA ophthalmic emulsion 0.05% was instilled in the right eyes of pigmented rabbits, in either a single or repeated quater in die (QID) for 3 days or 7 days) dose of 25 µg/50 µL. The radioactivity in right and left ocular tissues, urine, blood, plasma, and feces were measured with a liquid scintillation counter. Additionally, the distribution of radioactivity around ocular tissues was investigated with autoradiography. RESULTS: After a single instillation, the highest maximum radioactive concentrations were found in the cornea (2,081 ng eq./g), followed by the iris/ciliary body (926 ng eq./g), conjunctiva (330 ng eq./g), anterior retina/choroid (273 ng eq./g), sclera (222 ng eq./g), and aqueous humor (144 ng eq./mL) of treated eyes. The maximum radioactivity concentration of the posterior retina/choroid was 59 ng eq./g, and difluprednate delivered as a topical ophthalmic emulsion reached the back of the eye. However, radioactivity in untreated eyes was very low. Total radioactivity excreted in urine and feces 168 h after a single instillation was 99.5% of the total dose. Radioactivity concentration levels measured 24 h after 28 instillations were no more than twice those measured 24 h after 12 instillations. Radioactive concentrations in ocular and periocular tissues were highest at 0.5 or 1 h after a single instillation, and were mostly eliminated from these tissues by the end of the study. Radioactivity was barely detectable in the blood, with very little accumulation even after multiple doses. CONCLUSIONS: After instillation of (3)H-DFBA ophthalmic emulsion 0.05% in rabbit eyes, radioactivity was distributed at the anterior segment and cleared rapidly. Some radioactivity was detected in the posterior retina/choroid, suggesting that difluprednate and its metabolites reach these tissues. These results suggest that difluprednate delivered as a topical ophthalmic emulsion reached the anterior and posterior segments of the eye quickly and may be a potential treatment for ocular inflammation in these areas.

Metabolic profiles of difluprednate in rabbit ocular tissues after instillation of difluprednate ophthalmic emulsion.

Difluprednate (DFBA; 6alpha,9-difluoro-11beta,17,21-trihydroxy-1,4-pregnadiene-3,20-dione 21-acetate 17-butyrate) is an effective anti-inflammatory agent derived from prednisolone. The present study aimed to evaluate the metabolite profile of DFBA in rabbit ocular tissues after instillation of DFBA ophthalmic emulsion. The high-performance liquid chromatography with radiochemical detection was employed to analyze radioactivity in rabbit ocular tissues that had been instilled with a (3)H-DFBA 0.05% ophthalmic emulsion. At 0.5 and 2 h after instillation, DFBA was not detected in the cornea, aqueous humor, or iris/ciliary body. Instead, 21-deacetylated DFBA (DFB), 17-debutylated DFB (DF), and an unknown metabolite were found. The unknown metabolite was identified as de-17-side chain-glucocorticoid metabolite (DF21C), which is a novel glucocorticoid metabolite formed by the scission of the 17-side chain via an unknown metabolic reaction pathway. The K(i) value of DF21C was 5.6 x 10(-7) mol/L, indicating very weak glucocorticoid receptor binding of DF21C (approximately 1000-fold less than that of DFBA and DFB).

Bepotastine besilate, a highly selective histamine H(1) receptor antagonist, suppresses vascular hyperpermeability and eosinophil recruitment in in vitro and in vivo experimental allergic conjunctivitis models.

To elucidate the ocular pharmacological properties of bepotastine besilate, a selective histamine H(1) receptor antagonist, when compared with other histamine H(1) receptor antagonists, using guinea pig allergic conjunctivitis models and in vitro models of eosinophil recruitment and mast cell membrane stabilization. Conjunctival vascular hyperpermeability was studied in guinea pigs passively sensitized with anti-ovalbumin antiserum or following subconjunctival injection of histamine. Modulation of eosinophil recruitment was evaluated for both platelet-activating factor (PAF)-induced eosinophil infiltration in guinea pigs and leukotriene B(4)-induced in vitro chemotaxis of guinea pig peritoneal eosinophils. Membrane-stabilizing effects of bepotastine also were studied with rat peritoneal mast cells stimulated with the ionophore A23187. Histamine H(1) receptor antagonists including bepotastine besilate were topically administered before ovalbumin, histamine or PAF challenges for in vivo experiments or were added directly to mast cell and eosinophil medium in vitro. Bepotastine besilate significantly inhibited conjunctival vascular hyperpermeability in a dose-dependent manner with maximal effect for bepotastine besilate 1.5%. In separate in vivo experiments, bepotastine besilate 1.0% was significantly more effective than levocabastine 0.025% in the passive sensitization model or olopatadine 0.1% in the histamine-induced hyperpermeability model. Bepotastine besilate 1.0% further suppressed PAF-induced eosinophil infiltration into conjunctival tissue more effectively than ketotifen 0.05%. Chemotaxis of guinea pig peritoneal eosinophils and histamine release from rat peritoneal mast cells in vitro were also inhibited by addition of bepotastine. Olopatadine had a weak effect as compared to that of bepotastine on eosinophil chemotaxis and no effect on mast cell histamine release in our study conditions. Bepotastine besilate was more potent than olopatadine, ketotifen, or levocabastine in reducing vascular hyperpermeability in various animal models of allergic conjunctivitis. Mast cell function and eosinophil chemotaxis were also inhibited in vitro with bepotastine, suggesting bepotastine acts as an inhibitor of allergic response through multiple mechanisms: histamine H(1) receptor antagonism, mast cell stabilization, and inhibition of eosinophil migration to ocular inflammatory sites.

Effect of dosing interval on the efficacy of topical ophthalmic gatifloxacin against Enterococcus faecalis in an in vitro pharmacokinetic model simulating the local eye compartment.

Improved dosing regimens have been proposed for various antimicrobial agents by application of pharmacokinetic/pharmacodynamic (PK/PD) principles. However, for topical ophthalmic use there are several limitations to changing the dosing regimen, such as drug formulation and bioavailability. In this study, we investigated the relationship between dosing interval and antibacterial efficacy in an in vitro PK model mimicking post-operative endophthalmitis. The in vitro PK model simulated the aqueous humour concentration following topical application of 0.3% gatifloxacin ophthalmic solution to rabbit eyes. A clinical isolate of Enterococcus faecalis was exposed to gatifloxacin three times repeatedly at various intervals from 0 h to 8 h. The area between the control growth curve and the bacterial killing and re-growth curve for 24 h (ABBC) was used to evaluate efficacy. The ABBC showed bell-shaped dependence on the dosing interval with a peak at 3h. Under limited condition of total exposure amount, i.e. area under the concentration-time curve, the antimicrobial efficacy appears to be associated with the cumulative time of a 24-h period such that the concentration exceeds the minimum inhibitory concentration (T>MIC) rather than the peak concentration:MIC ratio. The length of intermission of T>MIC during repeated dosing appears to be proportional to the decrease in efficacy of gatifloxacin against E. faecalis. A longer dosing interval, as long as T>MIC is continuous, would likely be more efficient at preventing post-operative enterococcal endophthalmitis. However, further investigation is necessary to explore whether this model is applicable to a variety of pathogens and drugs.

Prophylactic efficacy of ophthalmic quinolones in experimental endophthalmitis in rabbits.

PURPOSE: The aim of this study was to determine the efficacy of 0.3% gatifloxacin ophthalmic solution in preventing bacterial endophthalmitis in rabbits. METHODS: Eighty-four (84) albino phakic rabbits were injected unilaterally with 2 x 10(4) colony forming units of Enterococcus faecalis into the anterior chamber. The eyes received 0.3% ofloxacin ophthalmic ointment or 0.3% gatifloxacin ophthalmic solution with different regimens in three separate experiments: (1) 1 or 3 drops of gatifloxacin every 2 h or a single application of ofloxacin for 1 day; (2) 3 drops/day of gatifloxacin application started at 0, 6, and 24 h postinoculation, or 1 drop at 0 h, and 3 times daily gatifloxacin for the following 3 days; and (3) 1 or 3 drops of gatifloxacin application started at 0 h and no further application for the following 3 days. The control eyes received no treatment in the three experiments. The effectiveness of these different regimens was assessed by slit-lamp biomicroscopy and bacterial colony counts. The ocular penetration of the drugs was determined in a separate experiment, using 36 normal albino rabbits. RESULTS: The concentration-time curves for gatifloxacin and ofloxacin appeared parallel, with mean peak concentrations of 1161 and 219 ng/mL, respectively, at 1 h postinstillation. In Experiment 1, gatifloxacin significantly reduced the inflammation and the number of living bacteria in the aqueous humor, compared with controls, whereas ofloxacin ointment did not. A single application of ofloxacin ointment was not better than 1 drop of gatifloxacin. The results of Experiment 2 showed that the effectiveness of gatifloxacin decreased as the interval between the inoculation and the onset of treatment increased. In Experiment 3, only 3 drops of gatifloxacin on day 1 kept the inflammation significantly lower than that in the control for 4 days. CONCLUSIONS: Immediate postoperative prophylaxis would likely be effective in reducing the risk of enterococcal endophthalmitis by topical gatifloxacin.

Immunomodulatory effect of gatifloxacin on mouse peritoneal macrophages in vitro and in models of endotoxin-induced rat conjunctivitis and rabbit bacterial keratitis.

AIM: To determine the anti-inflammatory activity of gatifloxacin in ophthalmic use. METHODS: The following 3 experiments were carried out. (1) Rabbits were inoculated intracorneally with methicillin-resistant Staphylococcus aureus and topically treated with gatifloxacin or levofloxacin. The severity of infection and viable bacterial count were assessed. (2) Thioglycollate-elicited mouse peritoneal macrophages were stimulated by Pseudomonas lipopolysaccharides (LPS) in the presence of graded concentrations of fluoroquinolones, and macrophage-derived tumor necrosis factor alpha (TNF-alpha) was assessed. (3) The effects of fluoroquinolones on TNF-alpha production were compared in an LPS-induced rat conjunctivitis model. RESULTS: In the rabbit keratitis model, the ocular inflammation was significantly reduced by gatifloxacin as compared to levofloxacin but there was no significant difference between the groups in the number of viable bacteria. Gatifloxacin and levofloxacin suppressed TNF-alpha production in mouse macrophages in a concentration-dependent manner, and the effect of gatifloxacin was more potent than that of levofloxacin. Moxifloxacin exhibited no effect in this condition. In the rat conjunctivitis model, the tissue TNF-alpha level was significantly reduced only in the group instilled with gatifloxacin ophthalmic solution. CONCLUSION: These results indicate that gatifloxacin has not only antibacterial activity but an anti-inflammatory action caused by at least inhibiting TNF-alpha production at the doses used in topical ophthalmic therapy.

Culture characteristics of carotenoid-producing filamentous fungus T-1, and carotenoid production.

The culture characteristics, carotenoid production, and associated biosynthetic pathway of strain T-1 were examined. As a result of examining the culture temperature and light irradiation, an increase of neurosporaxanthin and neurosporaxanthin beta-D-glucopyranoside was observed at a low temperature and 0 lx. It was suggested that highly polar carotenoids, such as neurosporaxanthin, and carotenoid glycosides were involved in the stabilization of membrane during nutrition storage other than the defense function of fungus bodies. Strain T-1 produced lycopene, beta-carotene, gamma-carotene, torulene, neurosporaxanthin, and neurosporaxanthin beta-D-glucopyranoside, as assessed by HPLC, LC-MS, and NMR analysis. Carotenoid biosynthesis begins with neurosporene, passing to lycopene and gamma-carotene through cyclization, and produces beta-carotene. In addition, it is saturated, gamma-carotene is converted to torulene, and neurosporaxanthin is produced. Thus, the carotenoid biosynthetic pathway in strain T-1 was estimated.

Effects of topical administration of y-39983, a selective rho-associated protein kinase inhibitor, on ocular tissues in rabbits and monkeys.

PURPOSE: To elucidate the intraocular pressure (IOP)-lowering effects and associated characteristics of Y-39983, a selective Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor derived from Y-27632, in animal eyes. METHODS: Y-39983 was compared with Y-27632 for selectivity of ROCK inhibition by biochemical assay. The IOP was monitored by pneumatonometer in albino rabbits and cynomolgus monkeys that were given topically administered Y-39983. The total outflow facility and uveoscleral outflow were measured by two-level constant-pressure perfusion and perfusion technique using fluorescein isothiocyanate-dextran, respectively, at 2 hours after topical administration of Y-39983 in albino rabbits. The ocular toxicologic effects of topical administration of Y-39983 were observed in albino rabbits and cynomolgus monkeys. RESULTS: A biochemical assay showed that Y-39983 inhibited ROCK more potently than Y-27632. In rabbits, topical administration of Y-39983 significantly increased conventional outflow by 65.5%, followed by significant, dose-dependent reduction in IOP. Maximum IOP reduction was 13.2 +/- 0.6 mm Hg (mean +/- SE) at 0.1% Y-39983 in rabbits. In monkeys, at 3 hours after topical administration of 0.05% Y-39983, maximum reduction of IOP was 2.5 +/- 0.8 mm Hg. No serious side effects were observed in ocular tissues except sporadic punctate subconjunctival hemorrhage during long-term topical administration of Y-39983 four times a day (at 2-hour intervals) in rabbits or monkeys. However, punctate subconjunctival hemorrhage was not observed with administration twice daily (at a 6-hour interval) or three times a day (at 5-hour intervals). CONCLUSIONS: Y-39983 causes increased outflow facility followed by IOP reduction. Y-39983 ophthalmic solution may be a candidate drug for lowering of IOP, since it increases conventional outflow and produces relatively few side effects.

Treatment of rabbit corneal infections with ophthalmic gatifloxacin: a concentration dependence study.

This study was designed to determine the most effective dose of gatifloxacin in ophthalmic solution for control of methicillin-resistant Staphylococcus aureus (MRSA) corneal infections in rabbits. Rabbits were inoculated by injecting 9300 colony-forming units of MRSA into the corneal stroma of the eye (n=43). They were then randomly assigned to topical administration of saline, ofloxacin 0.3%, or gatifloxacin 0.02%, 0.1%, 0.3%, or 0.5% ophthalmic solutions. Infection severity 48 hours postinoculation was assessed by masked observers using standard scales. After treatment completion, viable MRSA in corneal tissue were counted, and pathologic examinations of ocular tissues were conducted. Relative to saline, treatment with gatifloxacin 0.3% or 0.5% decreased mean infection scores at every time point from 16 to 48 hours after inoculation (P < or = .012) and reduced area-under-the-curve values for infection scores by 50.3% and 54.2%, respectively (P = .00005). Rabbits treated with gatifloxacin 0.3% and 0.5% had lower area-under-the-curve values than those treated with ofloxacin 0.3% (P < or = .039). Viable MRSA in corneal tissue after gatifloxacin 0.3% or 0.5% treatment were decreased to less than 1% of those found after ofloxacin 0.3% treatment. Gram-positive colony formation and abscesses found in saline-treated corneas were distinctly alleviated by treatment with gatifloxacin 0.3% or 0.5%. No significant differences were observed between treatments with gatifloxacin 0.3% or 0.5% ophthalmic formulations and they were equally effective. Topical administration of gatifloxacin 0.3% or 0.5% ophthalmic solutions controlled MRSA corneal infections in rabbits significantly better than saline or ofloxacin 0.3%.

Iganidipine, a new water-soluble Ca2+ antagonist: ocular and periocular penetration after instillation.

PURPOSE: To evaluate the potential of topical iganidipine ophthalmic solution to exert Ca(2+)-antagonistic activity in the posterior parts of the eye without inducing systemic effects, ocular and periocular penetration of topically instilled iganidipine was studied in pigmented rabbits. METHOD: First, (14)C-iganidipine solution (0.03%, 30 microL) was instilled into one eye, and vehicle into the other eye to determine the intraocular penetration of iganidipine and to measure the radioactivity of ocular tissues 0.25, 0.5, 1, 2, 4, and 12 hours after a single instillation (n = 3, respectively). Second, iganidipine (0.03%) or betaxolol (0.5%) was unilaterally instilled twice daily for 20 days to study the effects on intravitreously injected various doses of endothelin (ET)-1-induced retinal artery constriction to evaluate whether a pharmacologically active level of the drug penetrated to the posterior retina and to estimate the drug level in the posterior retina (n = 6, respectively). Third, iganidipine (0, 10, or 30 microg/kg: n = 6, 3, and 6, respectively) was intravenously injected to study the effects on intravitreously injected ET-1-induced retinal artery constriction to evaluate iganidipine levels in the posterior retina. Fourth, periocular penetration of iganidipine was studied by means of whole-head autoradiography after a single instillation of (14)C-iganidipine (0.09%, 30 microL; n = 5). RESULTS: Penetration of topically applied iganidipine to the cornea or aqueous humor was high and estimated to be at least 10 times higher than that reported for timolol or carteolol. Concentrations in the iris-ciliary body or retina-choroid were much higher than in the plasma, both in the treated and control eyes, suggesting that iganidipine binds to uveal pigments. Twice-daily 20-day instillation of iganidipine (0.03%), but not of betaxolol (0.5%), significantly suppressed constriction of the retinal arteries induced by intravitreous injection of ET-1 at a dose of 2.5 or 0.5 ng in the ipsilateral eye. Intravenous injection of iganidipine at a dose of 30 microg/kg (giving a free plasma concentration of approximately 10(-8) M), but not at a dose of 10 microg/kg, significantly suppressed intravitreous ET-1-induced (0.5 ng) constriction of the retinal artery to a similar degree as twice-daily 20-day instillation of 0.03% iganidipine. After a single instillation of 0.09% iganidipine, the equivalent concentration of iganidipine in the ipsilateral retrobulbar periocular space estimated from autoradiography was approximately 3.9 x 10(-8) M between 15 minutes and 1 hour after instillation, consistently higher than in the untreated contralateral eyes by approximately 3.0 x 10(-8) M (P = 0.043). CONCLUSIONS: In rabbits, topically instilled iganidipine, a Ca(2+) antagonist, in a 0.03% solution reaches the ipsilateral posterior retina or retrobulbar periocular space by local penetration at concentrations sufficient to act as a Ca(2+) antagonist.

A new carotenoid glycosyl ester isolated from a marine microorganism, Fusarium strain T-1.

A new carotenoid glycosyl ester, neurosporaxanthin beta-D-glucopyranoside (2), together with neurosporaxanthin (1), beta-carotene, gamma-carotene, and torulene were isolated from cultured cells of a marine microorganism, strain T-1, which was identified as Fusarium sp. Their structures were determined by chemical and spectral data.