A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing.

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay.

Real-Time PCR Assay for the Diagnosis and Quantification of Co-infections by Diaporthe batatas and Diaporthe destruens in Sweet Potato.

Foot rot disease caused by Diaporthe destruens (formerly Plenodomus destruens) has become a major concern for the production of sweet potato [Ipomoea batatas (L.) Lam.] in Japan. A related fungus Diaporthe batatas, which causes dry rot disease of sweet potato, is native and is widespread in fields in Japan. The similar characteristics of these two pathogens pose a challenge for conventional disease diagnosis. Currently, there are no effective molecular measures for identifying and distinguishing D. destruens and D. batatas. Here, we demonstrate a real-time PCR assay that distinguishes and quantifies D. batatas and D. destruens from co-infected sweet potato. The assay was performed with various simulated DNA combinations of D. batatas and D. destruens ranging from 1:1 to 1:100000. The assay was also used with the ratios of D. batatas: D. destruens: sweet potato DNA ranging from 1:1:1 to 1:1:100000. These assays produced a specific amplification product for each of the pathogens, and quantified the fungal biomass over the entire range tested without detecting false positives. The assay was validated by using infected sweet potato collected from various fields; it showed sufficient sensitivity and specificity to quantify and distinguish D. batatas and D. destruens from these field samples. Thus, our real-time PCR assay would be a useful tool for diagnosis of D. batatas and D. destruens and is expected to provide the foundation for the design of integrated disease management strategies for foot rot disease in sweet potato.

Chemoenzymatic semisynthesis of caffeic acid ß-phenethyl ester, an antioxidative component in propolis, from raw coffee bean extract.

Caffeic acid ß-phenethyl ester (CAPE), an antioxidative bioactive catechol isolated from propolis, was semisynthesized from chlorogenic acid and related compounds in an extract of raw (unroasted) Robusta coffee (Coffea canephora) beans in 5 steps and a total yield of 31%. Oxidative degradation of the intermediates and target molecule was prevented by alkaline hydrolysis of the chlorogenic acids in the presence of sodium dithionite (Na2S2O4) and deprotection of the catecholic diacetate precursor by Candida antarctica lipase B-mediated transesterification as the final step.

Real-Time PCR Detection of the Onion Downy Mildew Pathogen Peronospora destructor From Symptomless Onion Seedlings and Soils.

An outbreak of downy mildew disease of onion, caused by Peronospora destructor, in Japan in 2016 necessitated a reevaluation of the primary inoculum sources to optimize disease management. Detection of the P. destructor pathogen in plants with asymptomatic infection and in soil would guide the application of fungicides according to the extent of infection before disease development. Here, we detected P. destructor in both plants and soil using newly developed primer sets (Pd ITS and Pd ITS 614) by both conventional and real-time PCR. Validation by real-time PCR with Pd ITS 614 showed that P. destructor DNA was amplified from symptomless seedlings at 3.7 × 102 to 1.0 × 100 conidium cells/50 mg leaf tissue, suggesting the detection of asymptomatic infection. Real-time PCR with Pd ITS amplified pathogen DNA from field soils at 1.6 × 103 to 8.3 × 101 oospore cells/g of soil. This real-time PCR assay provides a useful tool for identifying and quantifying inoculum sources, which may be the foundation of the design of integrated disease management strategies.

A Novel HIF Inhibitor Halofuginone Prevents Neurodegeneration in a Murine Model of Retinal Ischemia-Reperfusion.

Neurodegeneration caused with retinal ischemia or high intraocular pressure is irreversible in general. We have focused on the role of hypoxia-inducible factor (HIF) in retinal homeostasis and revealed that HIF inhibition may be effective against retinal neovascular and neurodegeneration. In this study, we performed in vitro screening of natural products and found halofuginone, which is a derivative of febrifugine extracted from hydrangea, as a novel HIF inhibitor. Administration of halofuginone showed a significant neuroprotective effect by inhibiting HIF-1α expression in a murine retinal ischemia-reperfusion model histologically and functionally. These results indicate that halofuginone can be a neuroprotective agent in ischemic retinal degenerative diseases.

Integral role of receptor for advanced glycation end products (RAGE) in nondiabetic atherosclerosis.

An advanced glycation end products (AGE)/a receptor for AGE (RAGE) axis plays a central role in the pathogenesis of diabetic vascular remodeling. This study was conducted to clarify the role of RAGE in nondiabetic atherosclerosis. We used the aortic and coronary atherosclerotic lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits prone to myocardial infarction (WHHLMI) at 1 to 14 months. Immunohistochemistry demonstrated the significant expression of RAGE as early as at 1 month with the stronger expression at 3 and 7 months, which was remarkably diminished at 14 months. RAGE expression was concordant with AGE accumulation. The major original sources of RAGE expression were macrophages and smooth muscle cells in addition to endothelial cells, and RAGE expression was distributed in the areas of phospholipid products, a component of oxidized LDL and nitrotyrosine. The concentrations of serum AGE did not alter significantly with aging. These findings suggested the expression of RAGE was induced by hyperlipidemia and oxidative stress independent of diabetes in WHHLMI rabbits. Additionally, our in vitro study showed that silencing of RAGE tended to attenuate oxidized-LDL-triggered PAI-1 expression in human cultured macrophages, as well as oxidized-LDL-induced tissue factor expression in peritoneal macrophages, suggesting a possible role of RAGE in prothrombogenic molecular regulation. In conclusion, the present study provides in vivo evidence that RAGE plays an integral role in the initiation and progression of nondiabetic atherosclerosis, suggesting that RAGE may be a novel target for treating not only diabetic but also nondiabetic vascular complications.

Development of a functional thyroid model based on an organoid culture system.

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid-like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRASQ61R) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer.

Synthesis of enantiomerically enriched drug precursors and an insect pheromone via reduction of ketones using commercially available carbonyl reductase screening kit "Chiralscreen® OH".

Commercially available "Chiralscreen® OH" starter kit containing five types of carbonyl reductases (E001, E007, E031, E039, and E078) was used for the reduction of several aromatic and aliphatic ketones to obtain enantiomerically enriched drug precursors and an insect pheromone. Almost stereochemically pure secondary alcohols, used in the synthesis of drugs such as (R)-rasagiline mesylate, (S)-rivastigmine, (R)-chlorphenesin carbamate, and (R)-mexiletine, and the insect pheromone (4S,5R)-sitophilure, were conveniently obtained. The enzymes worked well with ketones containing at least one non-bulky substituent at the carbonyl group. The diverse stereochemical preference of the above five carbonyl reductases was clarified.

Novel channel-mediated choline transport in cholinergic neurons of the mouse retina.

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X2 purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X2 purinoceptors acquire permeability to large cations, such as N-methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,ß-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X2 purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X2 purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X2 purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X2 purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

Selenium-binding lactoferrin is taken into corneal epithelial cells by a receptor and prevents corneal damage in dry eye model animals.

The ocular surface is strongly affected by oxidative stress, which causes many ocular diseases including dry eye. Previously, we showed that selenium compounds, e.g., selenoprotein P and Se-lactoferrin, were candidates for treatment of dry eye. This paper shows the efficacy of Se-lactoferrin for the treatment of dry eye compared with Diquas as a control drug using two dry eye models and incorporation of lactoferrin into corneal epithelial cells via lactoferrin receptors. We show the efficacy of Se-lactoferrin eye drops in the tobacco smoke exposure rat dry eye model and short-term rabbit dry eye model, although Diquas eye drops were only effective in the short-term rabbit dry eye model. These results indicate that Se-lactoferrin was useful in the oxidative stress-causing dry eye model. Se-lactoferrin was taken into corneal epithelium cells via lactoferrin receptors. We identified LRP1 as the lactoferrin receptor in the corneal epithelium involved in lactoferrin uptake. Se-lactoferrin eye drops did not irritate the ocular surface of rabbits. Se-lactoferrin was an excellent candidate for treatment of dry eye, reducing oxidative stress by a novel mechanism.

DNA aptamer raised against advanced glycation end products inhibits melanoma growth in nude mice.

Epidemiological studies have suggested that diabetes is associated with an increased risk of cancer. However, the underlying molecular mechanism remains unclear. We investigated here whether DNA aptamer directed against advanced glycation end products (AGE-aptamer) inhibited melanoma growth in nude mice. G361 melanoma cells were injected intradermally into the upper flank of athymic nude mice. Mice received continuous intraperitoneal infusion (0.136 µg/day) of either AGE-aptamer (n=9) or Control-aptamer (n=8) by an osmotic mini pump. Tumor volume was measured at 4-day interval, and G361 melanoma was excised at day 43 after the aptamer treatment. We further examined the effects of AGE-aptamer on proliferation of AGE-exposed endothelial cells and G361 cells. AGE-aptamer significantly inhibited the in vivo-tumor growth of G361 melanoma. Immunohistochemical and western blotting analyses of G361 melanoma revealed that AGE-aptamer decreased expression levels of proliferating nuclear antigen, CD31 and Mac-3, markers of endothelial cells and macrophages, respectively. AGE-aptamer significantly decreased the number of tumor-associated vessels. AGE, receptor for AGE (RAGE) and vascular endothelial growth factor levels were also reduced in AGE-aptamer-treated G361 melanoma. AGE-aptamer inhibited the AGE-induced proliferation and tube formation of endothelial cells as well as the growth of G361 cells in vitro. The present findings suggest that AGE-aptamer could inhibit the AGE-RAGE axis in G361 melanoma and resultantly suppress the tumor growth in nude mice by blocking the angiogenesis. AGE-aptamer might be a novel therapeutic strategy for preventing the progression of malignant melanoma in diabetes.

[What should the radiation education in Japan in the future be like?].

In respect to policy and involvement in social cognition of Advanced Science and Technology, people desire to recognize the scientific understanding and social understanding hierarchically and simultaneously. However, the understandings of some sciences and technologies are dependent on the amount of information given and how easy it is to understand it. Nuclear power and radiation are a typical example of such sciences and technologies because their advantages and disadvantages are clear. On the other hand, the Fukushima Nuclear Plant Accident that occurred in March 2011 caused the myth about the safety and security of nuclear power to collapse. Concerns about nuclear power and radiation increased abruptly after the accident. Also the scientific understanding of 'nuclear power' and radiation increased. The content and level of radiation education was highly significant than before the accident. However, it is essential to propose a more detailed explanation for people that are concerned about radioactive contamination of food and also for people living in areas that still have relatively high dose of radioactive material. Although some technical problems such as the influences on the human body by low-dose exposure remain unresolved, not only specialists on nuclear power and radiation, but also the persons that have studied the radiation are desired to explain radiation for familiar people. As a result, in Japan, the learning of individuals spread to society because the Japanese are highly interested in nuclear power and radiation and the understanding of historical background.

Blockade by phosphorothioate aptamers of advanced glycation end products-induced damage in cultured pericytes and endothelial cells.

Advanced glycation end products (AGEs) not only inhibit DNA synthesis of retinal pericytes, but also elicit vascular hyperpermeability, pathological angiogenesis, and thrombogenic reactions by inducing vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) through the interaction with the receptor for AGEs (RAGE), thereby being involved in the pathogenesis of diabetic retinopathy. In this study, we screened novel phosphorothioate-modified aptamers directed against AGEs (AGEs-thioaptamers) using a combinatorial chemistry in vitro, and examined whether these aptamers could inhibit the AGE-induced damage in both retinal pericytes and human umbilical vein endothelial cells (HUVECs). We identified 11 AGEs-thioaptamers; among them, clones #4, #7s and #9s aptamers had higher binding affinity to AGEs-human serum albumin (HSA) than the others. Surface plasmon resonance analysis revealed that KD values of #4s, #7s and #9s were 0.63, 0.36, and 0.57nM, respectively. Furthermore, these 3 clones dose-dependently restored the decrease in DNA synthesis in AGE-exposed pericytes. AGEs significantly increased RAGE, VEGF and PAI-1 mRNA levels in HUVEC, all of which were completely blocked by the treatment with 20nM clone #4s aptamer. Quartz crystal microbalance analysis confirmed that #4s aptamer dose-dependently inhibited the binding of AGEs-HSA to RAGE. Our present study demonstrated that AGEs-thioaptamers could inhibit the harmful effects of AGEs in pericytes and HUVEC by suppressing the binding of AGEs to RAGE. Blockade by AGEs-thioaptamers of the AGEs-RAGE axis might be a novel therapeutic strategy for diabetic retinopathy.

DNA aptamer raised against AGEs blocks the progression of experimental diabetic nephropathy.

Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in diabetic nephropathy. We screened DNA aptamer directed against AGEs (AGEs-aptamer) in vitro and examined its effects on renal injury in KKAy/Ta mice, an animal model of type 2 diabetes. Eight-week-old male KKAy/Ta or C57BL/6J mice received continuous intraperitoneal infusion of AGEs- or control-aptamer for 8 weeks. AGEs-aptamer was detected and its level was increased in the kidney for at least 7 days. The elimination half-lives of AGEs-aptamer in the kidney were about 7 days. Compared with those in C57BL/6J mice, glomerular AGEs levels were significantly increased in KKAy/Ta mice, which were blocked by AGEs-aptamer. Urinary albumin and 8-hydroxy-2'-deoxy-guanosine levels were increased, and glomerular hypertrophy and enhanced extracellular matrix accumulation were observed in KKAy/Ta mice, all of which were prevented by AGEs-aptamer. Moreover, AGEs-aptamer significantly reduced gene expression of RAGE, monocyte chemoattractant protein-1, connective tissue growth factor, and type IV collagen both in the kidney of KKAy/Ta mice and in AGE-exposed human cultured mesangial cells. Our present data suggest that continuous administration of AGEs-aptamer could protect against experimental diabetic nephropathy by blocking the AGEs-RAGE axis and may be a feasible and promising therapeutic strategy for the treatment of diabetic nephropathy.

Epithelioid hemangioma (angiolymphoid hyperplasia with eosinophilia) in zosteriform distribution.

Epithelioid hemangioma (EH) or angiolymphoid hyperplasia with eosinophilia (ALHE) is an uncommon benign disease. We report an unusual case of EH (ALHE) that arose on the lower back in a zosteriform array. The presence of the characteristic histological appearance of plump endothelial cells with hobnail-like protrusions led to the diagnosis of EH (ALHE). Histological examination of the lesion also revealed the existence of arteriovenous shunts, the possible factor contributing to the pathogenesis of EH (ALHE).

Selenium compound protects corneal epithelium against oxidative stress.

The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.

Pigment epithelium-derived factor (PEDF) prevents platelet activation and aggregation in diabetic rats by blocking deleterious effects of advanced glycation end products (AGEs).

BACKGROUND: Alteration of platelet function contributes to microthrombus formation and may play an important role in the pathogenesis of diabetic vascular complications. In addition, there is a growing body of evidence that oxidative stress generation is involved in platelet activation and aggregation in vivo. Since we have recently found that pigment epithelium-derived factor (PEDF) inhibits thrombus formation in rats through its anti-oxidative properties, we investigated here whether PEDF prevented platelet activation and aggregation in diabetic or advanced glycation end products (AGEs)-injected rats. METHODS AND RESULTS: Experimental diabetes was induced by injecting streptozotocin to Sprague-Dawley rats. Diabetic or non-diabetic Sprague-Dawley rats were injected intravenously with or without 1 mg AGEs-bovine serum albumin or non-glycated bovine serum albumin in the presence or absence of 10 microg PEDF everyday. Administration of PEDF or pyridoxal phosphate, an inhibitor of AGEs formation, inhibited platelet P-selectin expression and aggregation by suppressing NADPH oxidase-driven superoxide generation, and subsequently ameliorated a shortened tail vein bleeding time in diabetic rats. Further, intravenous administration of AGEs to normal rats mimicked the effects of diabetes on platelet activation and bleeding time, which were also blocked by simultaneous administration of PEDF. CONCLUSIONS: These results demonstrated for the first time that PEDF inhibited platelet activation and aggregation in diabetic rats through its anti-oxidative properties. Our present study suggests that PEDF may play a protective role against diabetic vascular complications by attenuating the deleterious effects of AGEs on platelets.

Serum level of pigment epithelium derived factor (PEDF) is an independent determinant of resting heart rate in Japanese subjects.

We have recently found that serum level of pigment epithelium derived factor (PEDF) is significantly increased in proportion to the accumulation of the number of the components of the metabolic syndrome. Although high heart rate is also associated with metabolic risk factors for atherosclerosis, the correlation between serum level of PEDF and resting heart rate remains to be elucidated. In this study, we examined their relationship in Japanese outpatients. Four hundred two consecutive outpatients at Nakamura Clinic (141 male and 261 female; mean age 74.9+/-9.9) underwent a complete history and physical examination, determination of blood chemistries and PEDF levels. In multiple regression analysis, smoking (p=0.003), use of beta-blockers (p=0.010, inversely), presence of atrial fibrillation (p=0.014), PEDF (p=0.018) and glucose levels (p=0.033) were independently correlated to resting heart rate. Further, when mean serum PEDF levels were stratified by resting heart rates, a linear and significant trend (p=0.025) was observed. The present study demonstrated for the first time that serum level of PEDF was one of the independent determinants of resting heart rate in Japanese outpatients. PEDF may play some role in sympathetic nerve activity.

Serum levels of pigment epithelium-derived factor (PEDF) are positively associated with visceral adiposity in Japanese patients with type 2 diabetes.

BACKGROUND: Pigment epithelium-derived factor (PEDF) inhibits endothelial cell injury. Further, serum levels of PEDF are elevated in the metabolic syndrome. These observations suggest that PEDF may be elevated as a counter-system against vascular cell damage in the metabolic syndrome. However, little is known about the regulation of PEDF in patients with diabetes. In order to clarify the determinants of serum PEDF, here, we examined the relationship between the 1-year changes in PEDF levels and those in anthropometric and metabolic variables in type 2 diabetic patients. METHODS: Eighty-six consecutive outpatients with type 2 diabetes underwent a complete history and physical examination, determination of blood chemistries, and serum levels of PEDF at baseline and 1 year after. PEDF gene expression in cultured subcutaneous or omental adipocytes were analysed by quantitative real-time reverse transcription-polymerase chain reactions. RESULTS: Multiple regression analyses revealed that waist circumference, triglycerides, creatinine, and TNF-alpha were independently associated with PEDF. Further, the percent changes in serum levels of PEDF during 1-year observational periods were positively correlated with those of BMI. In addition, PEDF mRNA levels in cultured adipocytes were increased in parallel to the BMI values of subjects from whom adipocytes were derived, especially in omental adipocytes. CONCLUSION: These results demonstrated that serum levels of PEDF were positively associated with metabolic components and TNF-alpha in Japanese patients with type 2 diabetes. Our present study suggests that PEDF may be generated from adipose tissues and play some role in visceral obesity in type 2 diabetic patients.