Identification of a putative vacuolar serine protease gene in the rice blast fungus,Magnaporthe grisea

We identified and cloned a gene designated SPM1, encoding a serine protease from the rice blast fungus Magnaporthe grisea. SPM1 is a single-copy gene, encoding a subtilisin-like serine protease with 536 amino acids. Analyses of the deduced amino acid sequence of SPM1 suggested that SPM1 would be localized in a vacuole, an important organelle in pathogenicity.     

Regulated gene expression from adenovirus vectors: a systematic comparison of various inducible systems

Positively and tightly regulated gene expression is essential for gene function and gene therapy research. The currently-used inducible gene expression systems include tetracycline (Tet-on and T-REx), ecdysone, antiprogestin and dimerizer-based systems. Adenovirus (Ad) vectors play an important role in gene function and gene therapy research for their various advantages over other vector systems. Previously, we reported the inferiority of the Tet-on system as an inducible gene expression system in the context of Ad vectors in comparison with the Tet-off system. In this study, to identify an optimal system for regulated gene expression from Ad vectors, we made a rigorous direct comparison of these five inducible gene expression systems in three cell lines using the luciferase reporter gene. The highest sensitivity to the respective inducer was that of the dimerizer system, followed by the antiprogestin system. The lowest basal expression and the highest induction factor were both characteristic of the dimerizer system. Furthermore, the dimerizer and T-REx systems exhibited much higher induced expression levels than the other three systems. The elucidation of the characteristic features of each system should provide important information for widespread and feasible application of these systems. Overall, these results suggest the most appropriate inducible gene expression system in the context of Ad vectors to be the dimerizer system.     

Association between Psoriasis Vulgaris and Coronary Heart Disease in a Hospital-Based Population in Japan

BackgroundPsoriasis vulgaris is a chronic inflammatory skin disease with an immune-genetic background. It has been reported as an independent risk factor for coronary heart disease (CHD) in the United States and Europe. The purpose of this study was to investigate the association between psoriasis and CHD in a hospital-based population in Japan.MethodsFor 113,065 in-hospital and clinic patients at our institution between January 1, 2011 and January 1, 2013, the diagnostic International Classification of Diseases (ICD)-10 codes for CHD, hypertension, dyslipidemia, diabetes, and psoriasis vulgaris were extracted using the medical accounting system and electronic medical record, and were analyzed.ResultsThe prevalence of CHD (n = 5,167, 4.5%), hypertension (n = 16,476, 14.5%), dyslipidemia (n = 9,236, 8.1%), diabetes mellitus (n = 11,555, 10.2%), and psoriasis vulgaris (n = 1,197, 1.1%) were identified. The prevalence of CHD in patients with hypertension, dyslipidemia, diabetes, and psoriasis vulgaris were 21.3%, 22.2%, 21.1%, and 9.0%, respectively. In 1,197 psoriasis patients, those with CHD were older, more likely to be male, and had more number of the diseases surveyed by ICD-10 codes. Multivariate analysis showed that psoriasis vulgaris was an independent associated factor for CHD (adjusted odds ratio [OR]: 1.27; 95% confidence interval [CI]: 1.01–1.58; p = 0.0404) along with hypertension (adjusted OR: 7.78; 95% CI: 7.25–8.36; p < 0.0001), dyslipidemia (adjusted OR: 2.35; 95% CI: 2.19–2.52; p < 0.0001), and diabetes (adjusted OR: 2.86; 95% CI: 2.67–3.06; p < 0.0001).ConclusionPsoriasis vulgaris was independently associated with CHD in a hospital-based population in Japan.     

Cooling Treatment Transiently Increases the Permeability of Brain Capillary Endothelial Cells Through Translocation of Claudin-5

The blood–brain-barrier (BBB) is formed by different cell types, of which brain microvascular endothelial cells are major structural constituents. The goal of this study was to examine the effects of cooling on the permeability of the BBB with reference to tight junction formation of brain microendothelial cells. The sensorimotor cortex above the dura mater in adult male Wistar rats was focally cooled to a temperature of 5 °C for 1 h, then immunostaining for immunoglobulin G (IgG) was performed to evaluate the permeability of the BBB. Permeability produced by cooling was also evaluated in cultured murine brain endothelial cells (bEnd3) based on measurement of trans-epithelial electric resistance (TEER). Immunocytochemistry and Western blotting of proteins associated with tight junctions in bEnd3 were performed to determine protein distribution before and after cooling. After focal cooling of the rat brain cortex, diffuse immunostaining for IgG was observed primarily around the small vasculature and in the extracellular spaces of parenchyma of the cortex. In cultured bEnd3, TEER significantly decreased during cooling (15 °C) and recovered to normal levels after rewarming to 37 °C. Immunocytochemistry and Western blotting showed that claudin-5, a critical regulatory protein for tight junctions, was translocated from the membrane to the cytoplasm after cooling in cultured bEnd3 cells. These results suggest that focal brain cooling may open the BBB transiently through an effect on tight junctions of brain microendothelial cells, and that therapeutically this approach may allow control of BBB function and drug delivery through the BBB.     

C-Peptide Level in Fasting Plasma and Pooled Urine Predicts HbA1c after Hospitalization in Patients with Type 2 Diabetes Mellitus

In this study, we investigate how measures of insulin secretion and other clinical information affect long-term glycemic control in patients with type 2 diabetes mellitus. Between October 2012 and June 2014, we monitored 202 diabetes patients who were admitted to the hospital of Asahi Life Foundation for glycemic control, as well as for training and education in diabetes management. We measured glycated hemoglobin (HbA1c) six months after discharge to assess disease management. In univariate analysis, fasting plasma C-peptide immunoreactivity (F-CPR) and pooled urine CPR (U-CPR) were significantly associated with HbA1c, in contrast to ΔCPR and C-peptide index (CPI). This association was strongly independent of most other patient variables. In exploratory factor analysis, five underlying factors, namely insulin resistance, aging, sex differences, insulin secretion, and glycemic control, represented patient characteristics. In particular, insulin secretion and resistance strongly influenced F-CPR, while insulin secretion affected U-CPR. In conclusion, the data indicate that among patients with type 2 diabetes mellitus, F-CPR and U-CPR may predict improved glycemic control six months after hospitalization.     

Fluvial transport of carbon along the river-to-ocean continuum and its potential impacts on a brackish water food web in the Iwaki River watershed, northern Japan

Riverine transport of dissolved inorganic carbon (DIC) from land to the ocean is an important carbon flux that influences the carbon budget at the watershed scale. However, the dynamics of DIC in an entire river network has remained unknown, especially in mountainous Japanese watersheds. We examined the effects of watershed land use and geology on the transports of inorganic carbon as well as weathered silica (Si) and calcium (Ca) in the Iwaki River system where agricultural and residential areas have developed in the middle and lower parts of the watershed. The concentration and stable carbon isotope ratios (δ¹³C) of DIC showed the longitudinal increase of ¹³C-depleted inorganic carbon along the river. As a result, most streams and rivers were supersaturated in dissolved CO₂ that will eventually be emitted to the atmosphere. The possible origin of ¹³C-depleted carbon is CO₂ derived from the decomposition of organic matter in agricultural and urban landscapes, as well as from in-stream respiration. In addition, agricultural and urban areas, respectively, exported the large amount of dissolved Si and Ca to the rivers, suggesting that CO₂ increased by respiration accelerates the chemical weathering of silicate and carbonate materials in soils, river sediments, and/or urban infrastructure. Furthermore, riverine bicarbonate flux is likely to enter shell carbonates of Corbicula japonica, an aragonitic bivalve, in the downstream brackish lake (Lake Jusan). These results revealed that the flux of DIC from the human-dominated watersheds is a key to understanding the carbon dynamics and food-web structure along the land-to-river-to-ocean continuum.     

Characterization of plant circadian rhythms by employing Arabidopsis cultured cells with bioluminescence reporters

Recent intensive studies have begun to shed light on the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana. During the course of these previous studies, the most powerful technique, elegantly adopted, was a real-time bioluminescence monitoring system of circadian rhythms in intact plants carrying a luciferase (LUC) fusion transgene. We previously demonstrated that Arabidopsis cultured cells also retain an ability to generate circadian rhythms, at least partly. To further improve the cultured cell system for studies on circadian rhythms, here we adopted a bioluminescence monitoring system by establishing the cell lines carrying appropriate reporter genes, namely, CCA1::LUC and APRR1::LUC, with which CCA1 (CIRCADIAN CLOCK-ASSOCIATED1) and APRR1 (or TOC1) (ARABIDOPSIS PSEUDO-RESPONSE REGULATORS1 or TIMING OF CAB EXPRESSION1) are believed to be the components of the central oscillator. We report the results that consistently supported the view that the established cell lines, equipped with such bioluminescence reporters, might provide us with an advantageous means to characterize the plant circadian clock.     

Myrmecophilous aphids produce cuticular hydrocarbons that resemble those of their tending ants

Aphid-tending ants protect aphids from natural enemies and collect honeydew secreted by the aphids. However, ants also often prey on the aphids they attend. Aphids, therefore, like social parasites of ants, may well have evolved chemical mimicry as an anti-predation strategy. In this study, we aimed to determine whether the aphid Stomaphis yanonis actively produces cuticular hydrocarbons (CHCs) that resemble those of the tending ant Lasius fuji. In the wild, ants put their CHCs on the aphids that they are tending, so in this study we analyzed “ant-free” aphids. Mature aphids that exuviated in the absence of ant attendance had almost all of the hydrocarbon components that the ants’ CHCs had. Moreover, hydrocarbons artificially applied to the aphids’ body surface were lost by exuviation. Taken together, these findings indicate that mature aphids actively produced ant-like CHCs, and they constitute the first documentation of a chemical resemblance between aphids and ants in a specific aphid–ant association.     

Studies on Continuous Enzyme Reactions

The kinetics of hydrolysis of acetyl-dl-methionine in DEAE-cellulose-aminoacylase (EC 3.5.1.14) column and DEAE-Sephadex-aminoacylase column was studied.

The rate of hydrolysis of substrate was shown to be dependent on the flow rate and independent to the dimension of the enzyme column. The rate of hydrolysis of the substrate was equal in cases of down-ward flow and of up-ward flow. The deteriorated aminoacylase columns by long period operation were reactivated by the recharge of aminoacylase to them. The continuous enzyme reaction using an aminoacylase column was superior to the batch enzyme reaction using native aminoacylase.

The enzymatic properties of the water-insoluble aminoacylase prepared by linking mold aminoacylase (EC 3.5.1.14) to DEAE-Sephadex were studied and compared with those of the native aminoacylase.

Optimum pH values for hydrolysis of several substrates by the DEAE-Sephadex-amino-acylase complex (DSA-complex) shifted about 0.5~1.5 pH units more to the acid side than those by the native enzyme. On the effects of metal ions and inhibitors, substrate specificity, optical specificity and kinetic constants, no marked difference was observed between the native enzyme and the DSA-complex. Heat stability, optimum temperature and resistance towards proteases were increased by conversion from the native form to the insoluble enzyme. It was also observed that the DSA-complex was activated by urea.     

Tryptophan Production by a Lipoic Acid Auxotroph of Enterobacter aerogenes Having Both Pyruvic Acid Productivity and High Tryptophanase Activity

A new process for tryptophan production was established using a lipoic acid auxotrophic mutant, Enterobacter aerogenes l-12, which has both pyruvic acid productivity and tryptophanase activity. The process consists of the production of pyruvic acid from glucose by the washed cells and the subsequent conversion of the acid to tryptophan by the tryptophanase itself in the presence of indole and NH₄C1.

To prepare washed cells of which the tryptophanase activity and the pyruvic acid productivity were both high, it was best to culture the strain in a medium containing 1 % Polypepton, 0.2 % glucose, 3 μg/1 dl-lipoic acid, 0.05 % l-tryptophan, and mineral salts. The optimum composition of the reaction mixture for the pyruvic acid production by the washed cells was established. Under these conditions, 17 g/1 of pyruvic acid was accumulated from 5 % glucose after 36 hr of incubation. Thus, the conversion of the pyruvic acid to tryptophan was done by adding indole, NH₄C1, pyridoxal-5′-phosphate, Triton X-100, and KOH to adjust the pH to 9.0 to the above reaction mixture. As a result, the pyruvic acid was rapidly converted to tryptophan, and the concentration of 14 g/1 was obtained after 36 hr (total 72 hr).