Ergot alkaloids in sclerotia collected in Japan: synthetic profiles and induction of apoptosis by Clavine-type compounds.

The genus Claviceps (Clavicipitaceae) is famous for producing ergot alkaloids (EAs) in sclerotia. EAs can cause ergotism, resulting in convulsions and necrosis when ingested, making these compounds a serious concern for food safety. Agroclavine (2), a typical Clavine-type EA, is a causative agent of ergotism and is listed as a compound to be monitored by the European Food Safety Authority. Clavine-type EAs are known to cause cytotoxicity, but the mechanism has not been elucidated. We performed annexin V and PI double-staining followed by flow cytometric analysis to detect apoptosis in HepG2 and PANC-1 cells after exposure to Clavine-type EAs. Clavine-type EAs reduced cell viability and induced apoptosis in both cell lines. We then performed LC-MS analysis of EAs from 41 sclerotia samples of Claviceps collected in Japan. 24 out of 41 sclerotia extracts include peptide-type EAs (ergosine/inine: 4/4', ergotamine: 5, ergocornine/inine: 6/6', α-ergocryptine/inine: 8/8', and ergocristine/inine: 9/9') and 19 sclerotia extracts among 24 sclerotia detected peptide type EAs include Clavine-type EAs (pyroclavine: 1, agroclavine: 2, festuclavine: 3) by LC-MS. We then performed a metabolomic analysis of the EAs in the sclerotia using principal component analysis (PCA). The PCA score plots calculated for EAs suggested the existence of four groups with different EA production patterns. One of the groups was formed by the contribution of Clavine-type EAs. These results suggest that Clavine-type EAs are a family of compounds requiring attention in food safety and livestock production in Japan.

Identification of a multi-component berberine 11-hydroxylase from Burkholderia sp. strain CJ1.

Berberine (BBR) is a protoberberine alkaloid extracted from plants such as Coptis japonica (Ranunculaceae). In a previous report, we demonstrated the existence of a 11-hydroxylation pathway employed by BBR-utilizing bacteria for metabolism of BBR. In the present study, we report the identification of the genes brhA, brhB, and brhC as encoding a multicomponent BBR 11-hydroxylase in Burkholderia sp. strain CJ1. BrhA is belonging to the Rieske non-heme iron oxygenase (RO) family, a class of enzymes known to catalyze the first step in bacterial aromatic-ring hydroxylation. We further demonstrate that BrhA activity requires BrhB (ferredoxin reductase) and BrhC (ferredoxin) as electron transport chain components. A BLAST search revealed that BrhA exhibits 38% and 33% sequence identity to dicamba O-demethylase (DdmC; AY786443) and chloroacetanilide herbicides N-dealkylase (CndA; KJ461679), respectively. To our knowledge, this work represents the first report of a bacterial oxygenase catalyzing the metabolism of a polycyclic aromatic-ring alkaloid.Abbreviations: BBR: berberine; D-BBR: demethyleneberberine; H-BBR: 11-hydroxyberberine; HD-BBR: 11-hydroxydemethyleneberberine; HDBA: 2-hydroxy-3,4-dimethoxybenzeneacetic acid; PAL: palmatine; H-PAL: 11-hydroxypalmatine; BRU: berberrubine; Fd: ferredoxin; FdR: ferredoxin reductase; ETC: electron transport chain.

Characterization of Burkholderia sp. strain CJ1, a newly isolated berberine-degrading bacterium from rhizosphere of Coptis japonica.

Burkholderia sp. strain CJ1 was newly isolated as berberine (BBR) degrading bacteria from rhizosphere of Coptis japonica. CJ1 had the ability to utilize BBR as the sole carbon source and revealed that BBR metabolism via 11-hydroxylation and demethylenation pathway. It was also revealed that the 11-hydroxylation ability of BBR and palmatine (PAL) has induced by BBR.

Antimicrobial agent isolated from Coptidis rhizome extract incubated with Rhodococcus sp. strain BD7100.

Coptidis rhizome (CR) is a widely used herbal medicine that contains protoberberine-type alkaloids. CR extract exhibits various pharmacologic activities. A previous study reported the isolation of Rhodococcus sp. strain BD7100 as a berberine (BBR)-utilizing bacterium, and the BBR-degradation pathway has been investigated. When we incubated strain BD7100 cells with CR extract, the number of viable cells declined with the degradation of components in the CR extract, and the culture broth exhibited antibacterial activity against strain BD7100. These results suggest that CR extract cultured in the presence of strain BD7100 contains one or more antibacterial agents. In this study, we isolated coptirhoquinone A (1) from CR extract incubated with strain BD7100 in Luria-Bertani (LB) medium, and the structure was elucidated using NMR and MS analysis. We also report the total synthesis and antimicrobial activities of 1 against bacteria, fungi, and Pythium sp.

Production of an emericellin and its analogues as fungal biological responses for Shimbu-to extract.

This research examined the production of fungal metabolites as a biological response to Kampo medicines. Shimbu-to (SMB) is a Kampo medicine composed of five herbal components: peony root (Shakuyaku), ginger (Shokyo), processed aconite root (Bushi), Poria sclerotium (Bukuryo), and Atractylodes lancea rhizomes (Sojutsu). High-performance liquid chromatography (HPLC) analysis of the fungus Aspergillus nidulans CBS 112.46 incubated in potato dextrose broth supplemented with SMB extract revealed emericellin (2) as the major peak and new xanthone analogues 24-hydroxyshamixanthone (1), shamixanthone (3), epishamixanthone (4), pre-shamixanthone (5), and variecoxanthone A (6) as minor peaks. The structure of 1 was determined by detailed analysis of 1D-NMR, 2D-NMR, and MS data. The results suggest that SMB extract regulates the biosynthesis of emericellin and its analogues in A. nidulans. Further investigations revealed that glucose induces the biosynthesis of emericellin and its analogues in A. nidulans in a concentration-dependent manner.

[Estimate of the Approximation Function of Spectral Hounsfield Unit Curves in Dual Energy CT Imaging].

The purpose of this study was to reveal the optimal function for regression of spectral Hounsfield Unit (HU) curves. The optimization procedure consists of the following steps: 1) obtaining dual energy CT (DECT) images of the RMI 467 phantom, 2) obtaining virtual monochromatic images from DECT images, 3) mapping each region of interest (ROI) to a phantom rod on virtual monochromatic images, 4) obtaining spectral HU curves for all rods, 5) regression of spectral HU curves using various functions, including linear, quadratic polynomial, cubic polynomial, quartic polynomial, quintic polynomial, sextic polynomial, septic polynomial, exponential, corrected exponential, bi-exponential, and logarithm, and 6) calculating the coefficients and the Akaike Information Criterion (AIC) of the functions listed above. Results indicated that the quintic polynomial function is suitable for analyzing the regression of spectral HU curves. The coefficients generated by the quartic or higher order polynomial functions were significantly higher than those generated by other functions (p<0.05). The median AIC of the quintic polynomial was the lowest among all functions. Therefore, we conclude that the quintic polynomial is the best function to use for the regression of spectral HU curves.

Intraocular pressure and visual field changes in normal-tension glaucoma patients treated using either unoprostone or latanoprost: a prospective comparative study.

PURPOSE: We conducted a prospective study in patients with normal-tension glaucoma (NTG) who received either isopropyl unoprostone or latanoprost. We compared the drugs in terms of their effects on intraocular pressure (IOP) and visual field loss progression over a 3-year period. STUDY DESIGN: Prospective, randomized controlled study. METHODS: We enrolled 48 patients with newly diagnosed NTG at Kanazawa University Hospital. Eligible patients were randomly allocated (1:1) to receive either unoprostone or latanoprost ophthalmic solutions. The primary outcomes were IOP changes and visual field deterioration within 36 months. Visual field changes were analyzed: the cumulative survival rates were calculated in terms of mean deviation, pattern standard deviation, and total deviation of the upper or lower hemi-visual field, each visual field sector, and guided progression analysis. In addition, we evaluated the progression of glaucomatous optic disc changes using fundus photography and confocal scanning laser ophthalmoscopy. RESULTS: The mean pretreatment IOP was 15.0±2.4 mmHg in the Unoprostone group and 15.2±1.9 mmHg in the Latanoprost group. The mean IOP during the treatment period was 13.7±2.3 mmHg in the Unoprostone group and 13.0±1.8 mmHg in the Latanoprost group. In both groups, the IOP decreased significantly (p<0.001) from baseline after treatment. The posttreatment IOP values were significantly lower in the Latanoprost group than in the Unoprostone group (p=0.023). Regarding the 3-year cumulative survival rate of visual field loss progression, there were no significant differences between groups in any parameters of the visual field or guided progression analysis. There were no significant differences between groups in disc changes. CONCLUSIONS: No significant differences were found between groups with regard to the visual field and structural progression in patients with NTG, although unoprostone was less effective than latanoprost in lowering the IOP.

Common origin of methylenedioxy ring degradation and demethylation in bacteria.

Plants produce many specific secondary metabolites as a response to environmental stress, especially biological stress. These compounds show strong biological activities and high stability against degradation by microbes and animals. Berberine, a benzylisoquinoline alkaloid, is found in many plant species and has strong antimicrobial activity, and is often included in traditional herbal medicines. We previously investigated how berberine is degraded in nature and we isolated two berberine-utilizing bacteria. In this study, we characterized the gene encoding the enzyme that degrades the 2,3-methylenedioxy ring of berberine; this ring is important for its activity and stability. Further characterization of several other berberine-utilizing bacteria and the genes encoding key demethylenation enzymes revealed that these enzymes are tetrahydrofolate dependent and similar to demethylation enzymes such as GcvT. Because the degradation of O-methyl groups or the methylenedioxy ring in phenolic compounds such as lignin, lignan and many other natural products, including berberine, is the key step for the catabolism of these compounds, our discovery reveals the common origin of the catabolism of these stable chemicals in bacteria.

Asnovolins A-G, Spiromeroterpenoids Isolated from the Fungus Aspergillus novofumigatus, and Suppression of Fibronectin Expression by Asnovolin E.

Seven novel spiromeroterpenoids, asnovolins A-G (1-7), one of which was shown to suppress fibronectin expression, were isolated from Aspergillus novofumigatus CBS117520 along with a known compound, novofumigatonin (8). The structures of asnovolins A-G were elucidated using MS and 2D-NMR data. Asnovolin E (5) suppressed fibronectin expression by normal human neonatal dermal fibroblast cells.

Isolation and identification of berberine and berberrubine metabolites by berberine-utilizing bacterium Rhodococcus sp. strain BD7100.

Based on the finding of a novel berberine (BBR)-utilizing bacterium, Rhodococcus sp. strain BD7100, we investigated the degradation of BBR and its analog berberrubine (BRU). Resting cells of BD7100 demethylenated BBR and BRU, yielding benzeneacetic acid analogs. Isolation of benzeneacetic acid analogs suggested that BD7100 degraded the isoquinoline ring of the protoberberine skeleton. This work represents the first report of cleavage of protoberberine skeleton by a microorganism.

Isolation of growth inhibitors of the snow rot pathogen Pythium iwayamai from an arctic strain of Trichoderma polysporum.

Growth inhibitors were isolated from an arctic strain of Trichoderma polysporum, and the structures were elucidated and the in vitro inhibitory effects of these compounds against Pythium iwayamai were investigated. Eleven compounds were isolated; four showed a concentration-dependent growth-inhibitory effect against P. iwayamai. None of these compounds have been reported previously as substances with antimicrobial activity against P. iwayamai. One of these four compounds inhibited the growth of the pathogen at 33 µg ml(-1) concentration during a 15-day incubation at 20 °C. This effect was comparable to that of chloroneb (1: 1,4-dichloro-2,5-dimethoxybenzene), a fungicide with activity against P. iwayamai. Thus, the results of the present study show that the arctic strain of T. polysporum can be an effective source of antibiotics with activity against the snow rot pathogen, P. iwayamai.

Isolation and structure elucidation of new phthalide and phthalane derivatives, isolated as antimicrobial agents from Emericella sp. IFM57991.

Three new phthalide derivatives, emefuranones A1, A2 and B (1-3); six new phthalane derivatives, emefuran A, B1, B2, C1, C2 and D (4-9); three new farnesylated phthalide derivatives, farnesylemefuranones A-C (10-12); xylarinol C (13); and emericelloxide (14), along with four known compounds (dustanin, sorbicillin, aspergillodiol and xylarinol A), were isolated from the culture extracts of Emericella sp. IFM57991. Structures of 1-14 were elucidated on the basis of spectroscopic analysis and chemical evidence. Compounds 4-7 and 13 showed moderate antibacterial activities against Bacillus subtilis.

11-Hydroxylation of Protoberberine by the Novel Berberine-Utilizing Aerobic Bacterium Sphingobium sp. Strain BD3100.

Protoberberine alkaloids, including berberine, palmatine, and berberrubine, are produced by medicinal plants and are known to have various pharmacological effects. We isolated two berberine-utilizing bacteria, Sphingobium sp. strain BD3100 and Rhodococcus sp. strain BD7100, from soil collected at a natural medicine factory. BD3100 had the unique ability to utilize berberine or palmatine as the sole carbon and energy source. BD3100 produced demethyleneberberine in berberine-supplemented medium. In a resting-cell incubation with berberine, BD3100 produced 11-hydroxyberberine; the structure of 11-hydroxyberberine was determined by detailed analysis of NMR and MS spectroscopic data. α-Naphthoflavone, miconazole, and ketoconazole, which are known inhibitors of cytochrome P450, interfered with BD3100 metabolism of berberine in resting cells. Inhibition by miconazole led to the production of a new compound, 11-hydroxydemethyleneberberine. In a resting-cell incubation with palmatine, BD3100 generated 11-hydroxypalmatine. This work represents the first report of the isolation and characterization of novel berberine-utilizing aerobic bacteria for the production of 11-hydroxylation derivatives of berberine and palmatine.

Association between genetic polymorphisms of the prostaglandin F2α receptor gene, and response to latanoprost in patients with glaucoma and ocular hypertension.

AIM: To examine whether intraocular pressure (IOP) reduction by latanoprost correlates with single nucleotide polymorphisms (SNPs) of the prostaglandin F2α (FP) receptor gene in patients with glaucoma and ocular hypertension (OH). METHODS: The genotype of nine SNPs in the FP receptor gene was determined by direct DNA sequencing, or other techniques, in 82 patients with glaucoma or OH who were treated with latanoprost monotherapy in one eye. The IOP reduction was evaluated by the percent IOP reduction (%ΔIOP), estimated by subtracting IOP fluctuations in the untreated fellow eye. Subjects were classified by %ΔIOP into low responders (%ΔIOP<10%) and others (%ΔIOP ≥10%). The correlation between %ΔIOP and SNPs in the FP receptor gene was analysed. RESULTS: Multiple regression analysis demonstrated that the rs12093097 was the only significant factor that correlated with %ΔIOP (p=0.039). Among estimated haplotypes, one haplotype that contained the minor allele only in rs3753380, was significantly correlated with low responders even after correction for multiple test (permutation test, p=0.037). CONCLUSIONS: An association was found between SNPs of the FP receptor gene and the response to latanoprost in patients with glaucoma or OH. The FP receptor genetic polymorphism may influence the degree of IOP reduction by latanoprost in these patients.

Effect of cataract and its removal on ganglion cell complex thickness and peripapillary retinal nerve fiber layer thickness measurements by fourier-domain optical coherence tomography.

PURPOSE: To examine the effect of cataract on signal strength index (SSI) and thickness measurements of the ganglion cell complex (GCC) and peripapillary retinal nerve fiber layer (pRNFL) with the RTVue-100 Fourier-domain optical coherence tomography device. METHOD: GCC and pRNFL measurements were obtained preoperatively in 53 eyes of 53 patients, including 13 glaucoma patients and measurements were repeated postoperatively. Cataract degree was graded by the Lens Opacities Classification System III. Factors associated with segmentation error and with changes in SSI or average thickness of GCC and pRNFL were determined. RESULTS: The prevalence and proportion of segmentation error were higher in GCC measurements compared with pRNFL measurements (prevalence: 28.3% vs. 5.6%, P<0.001; proportion: 5.9% vs. 1.3%, P=0.01). Multivariate analysis showed that segmentation error in GCC measurements was significantly associated with preoperative SSI and the degree of posterior subcapsular opacity. In eyes without segmentation error, postoperative SSI was significantly higher for GCC and pRNFL measurements, whereas postoperative thickness was slightly but significantly higher for all GCC and pRNFL parameters. Multivariate analysis indicated that preoperative SSI was highly correlated with SSI changes and that the presence of glaucoma and preoperative SSI were significantly correlated with average thickness changes of GCC and pRNFL, respectively. CONCLUSIONS: In eyes with cataract, segmentation error occurred more frequently in GCC measurements than in pRNFL measurements. After cataract surgery, all thickness parameters in GCC and pRNFL measurements increased slightly. Segmentation errors should be determined, especially in GCC measurements of eyes with cataract and low SSI.

Relationship between optic disc ovality and horizontal disc tilt in normal young subjects.

PURPOSE: To investigate the relationship between optic disc ovality and horizontal disc tilt. METHODS: The ovality index (the ratio of minimum to maximum disc diameter), horizontal and vertical optic disc tilt, and optic disc torsion were measured on the Heidelberg Retina Tomograph 3 printout in 381 right eyes of 381 normal young subjects. Stereophotographically obvious horizontal tilt and the relationship between the ovality index and horizontal tilt were determined in non-torted discs with an absolute value of disc torsion ≤15°. RESULTS: The median values of the ovality index, horizontal and vertical tilt, and disc torsion in all subjects were 0.92, 9.0°, 0.0°, and 0.0°, respectively. In the non-torted discs (205 eyes, 53.8 %), a horizontal tilt of ≥20° was stereoscopically obvious. The ovality index was significantly correlated with horizontal tilt (r = -0.276, P < 0.001). The area under the receiver operation curve for the ovality index to discriminate a disc with a horizontal tilt of ≥20° was 0.861. The horizontal disc tilt was significantly correlated with spherical equivalent (r = -0.369, P < 0.001) and axial length (r = 0.277, P < 0.001), but the ovality index was not. CONCLUSIONS: Although the ovality index may be an indicator of stereoscopically obvious horizontal disc tilt, horizontal tilt is more closely related with myopia.

Differentiation by imaging of superior segmental optic hypoplasia and normal-tension glaucoma with inferior visual field defects only.

PURPOSE: To differentiate superior segmental optic hypoplasia (SSOH) from normal-tension glaucoma (NTG) with inferior visual field defects only. METHODS: Eighteen eyes with SSOH (SSOH group) and 19 eyes with NTG (NTG group) were examined by optical coherence tomography (OCT), Heidelberg retina tomography (Heidelberg Retinal Tomograph II, HRT II) and standard automated perimetry. RESULTS: Retinal nerve fiber layer thickness (RNFLT) based on OCT measurements was significantly reduced (thinner) in the superior to superonasal sectors and significantly greater (thicker) in the inferotemporal sector in the SSOH group than in the NTG group. The cup was significantly smaller and the rim significantly larger in the superotemporal and temporal sectors in the SSOH group than in the NTG group based on HRT II measurements. The greatest area under the receiver operating characteristic curve for discrimination of SSOH from NTG by OCT and HRT II was for the RNFLT ratio of 1 + 2 o'clock/10 + 11 o'clock (0.985) and for the ratio of the superonasal to superotemporal sector of rim to disc area ratio and cup to disc area ratio (0.955), respectively. The frequent location of the inferior visual field defects corresponded to the difference in structural changes in both groups. CONCLUSIONS: Comparison of the superonasal to superotemporal sectors by OCT and HRT II were useful in differentiating SSOH from NTG with only inferior visual field defects.

Relationship between macular ganglion cell complex parameters and visual field parameters after tumor resection in chiasmal compression.

PURPOSE: To evaluate the relationship between macular ganglion cell complex (GCC) parameters and visual field (VF) parameters in chiasmal compression and the potential for GCC parameters in order to predict the short-term postsurgical VF. METHODS: Twenty-three eyes of 12 patients with chiasmal compression and 33 control eyes were studied. All patients underwent transsphenoidal tumor resection. Before surgery a 3D scan of the macula was taken using spectral-domain optical coherence tomography. All patients underwent Humphrey 24-2 VF testing after surgery. Spearman's rank correlation coefficients were used to evaluate the relationship between the GCC parameters and VF parameters [mean deviation (MD), pattern standard deviation]. Coefficients of determination (R2) were calculated using linear regression. RESULTS: Average thickness in the patients was significantly thinner than that of controls. Average thickness, global loss volume and focal loss volume (FLV) significantly correlated with the MD. We observed the greatest R2 between FLV and MD. CONCLUSIONS: Examining the macular GCC was useful for evaluating structural damage in patients with chiasmal compression. Preoperative GCC parameters, especially FLV, may be useful in predicting visual function following surgical decompression of chiasmal compression.

[Basic concept for the inspection of imported foods].

According to Article 4 of the Food Safety Basic Law (Law No. 48 of 2003), food safety must be ensured by taking necessary measures appropriately at each step of the food supply process both in Japan and overseas. From this viewpoint, for ensuring safety of imported foods further, the quarantine stations have obligations such as confirmation of the import-notification documents submitted under Article 27 of the Law, issue of inspection orders in accordance with Article 26 of the Law for imported foods suspected with high probability of violating the Act (in order to prevent harm to public health from the perspective of food sanitation), and implement of monitoring inspection under Article 28 of the Law to enforce checking of the food-sanitation conditions of various imported foods. If a quarantine station or local organization (including prefectures, cities and specially designated wards that operate public health centers) identify any violation of the Law, the Ministry of Health, Labour and Welfare (hereinafter referred to as "the MHLW"), the quarantine station and the related prefecture shall cooperate to take appropriate measures to help the corresponded importer recall the food as soon as possible. In addition, taking measures to prevent recurrence of such violations by demonstration of examples of violations and guidance to importers. Under the recent situation of accidents such as addition of toxic substances to the food, it is important to make continuous efforts for achieving food safety in exporting countries. With recognition that assurance of the safety of the consumer from foodborn illness, it is necessary for the MHLW, the Agency of Consumer Affaires, local governments and related offices to make active efforts together through risk evaluation, risk management and the risk communication for 1 while measuring the communication.

Evaluation of macular thickness and peripapillary retinal nerve fiber layer thickness for detection of early glaucoma using spectral domain optical coherence tomography.

PURPOSE: To evaluate the diagnostic ability of macular parameters and peripapillary retinal nerve fiber layer (RNFL) parameters for early glaucoma using spectral domain optical coherence tomography (SD-OCT). MATERIALS AND METHODS: One eye from 32 early glaucoma patients (including preperimetric glaucoma) and 32 normal participants underwent macular scans and peripapillary RNFL scans with SD-OCT 3 times on the same day. The discrimination power of each parameter to detect early glaucoma was determined by areas under receiver operating characteristics curve (AROC) and sensitivity at fixed specificity. Correlation of OCT data with visual field defects was evaluated by linear regression analysis. Reproducibility was also evaluated. RESULTS: Significant differences between early glaucoma and normal participants were found for all parameters except fovea in macular scans and in the superior and inferior quadrants, at 12, 3, 6, 7, 11 o'clock, and average RNFL thickness in RNFL scans. The best parameters based on AROC and sensitivity at a specificity of >90% were temporal outer macula thickness (AROC, 0.79; sensitivity, 63%) in macular parameters and inferior quadrant (AROC, 0.82; sensitivity, 53%) in RNFL parameters. The highest correlation with mean deviation was found in inferior inner macular volume (r=0.50, P<0.001). The mean intraclass correlation coefficient was 0.96 in macular scans and 0.84 in RNFL scans. Test-retest variability ranged from 2.3 to 10.1 µm in macular thickness, 0 to 0.06 mm in macular volume, and 5.8 to 18.9 µm in RNFL thickness. CONCLUSION: For the diagnosis of early glaucoma by SD-OCT, macular parameters had high discriminating power and high reproducibility comparable with peripapillary RNFL parameters.