Role of the prosthetic groups of NADPH-cytochrome P450 reductase in 3-hydroxyanthranilamide-catalyzed,NADPH-dependent superoxide anion production

Summary

The role of the prosthetic groups (FAD and FMN) of NADPH-cytochrome P450 reductase (P450 reductase)in 3-hydroxyanthranilamide (3-OH An.Amide)-catalyzed, NADPH-dependent superoxide anion (O₂-) production via the reductase was examined using the native and FMN-depleted preparations of P450 reductase which was partially purified from rat liver microsomes. NADPH-dependent O₂-production by the FMN-depleted preparation was about 10% of that by the native preparation. 3-OH An. Amide-catalyzed, NADPH-dependent O₂-production by the FMN-depleted preparation was less than 10% of that by the native preparation. FMN supplementation returned O₂-production to near normal. We observed the same results for NADPH oxidation and hydrogen peroxide formation. O₂-production, NADPH oxidation, and hydrogen peroxide formation were inhibited by native superoxide dismutase (SOD), but not by boiled, denatured SOD. These results indicate that the prosthetic groups, especially FMN, of P450 reductase play a critical role in 3-OH An.Amide-catalyzed, NADPH-dependent O₂-production via the reductase.     

Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray

We analyzed the urine samples of patients with type 2 diabetes at various stages of diabetic nephropathy by lectin microarray to identify a biomarker to predict the progression of diabetic nephropathy. Japanese patients with type 2 diabetes at various stages of nephropathy were enrolled and we performed lectin microarray analyses (n = 17) and measured urinary excretion of fetuin-A (n = 85). The increased signals of urine samples were observed in Siaα2-6Gal/GalNAc-binding lectins (SNA, SSA, TJA-I) during the progression of diabetic nephropathy. We next isolated sialylated glycoproteins by using SSA-lectin affinity chromatography and identified fetuin-A by liquid chromatography–tandem mass spectrometer. Urinary excretion of fetuin-A significantly increased during the progression of albuminuria (A1, 0.40±0.43; A2, 0.60±0.53; A3 1.57±1.13 ng/gCr; p = 7.29×10⁻⁸) and of GFR stages (G1, 0.39±0.39; G2, 0.49±0.45; G3, 1.25±1.18; G4, 1.34±0.80 ng/gCr; p = 3.89×10⁻⁴). Multivariate logistic regression analysis was employed to assess fetuin-A as a risk for diabetic nephropathy with microalbuminuria or GFR<60 mL/min. Fetuin-A is demonstrated as a risk factor for both microalbuminuria and reduction of GFR in diabetic nephropathy with the odds ratio of 4.721 (1.881–11.844) and 3.739 (1.785–7.841), respectively. Collectively, the glycan profiling analysis is useful method to identify the urine biomarkers and fetuin-A is a candidate to predict the progression of diabetic nephropathy.     

Metabolome analysis of food-chain between plants and insects

Evolution has shown the co-dependency between host plants and predators (insects), especially inevitable dependency of predators on plant biomass for securing their energy sources. It was postulated that NAD⁺ source used for major energy producing pathway is the glycerol-3-phosphate shuttle in insects. Using high throughput metabolomics approach, we found that larva of leaf beetle (Gastrophysa atrocyanea), which feed oxalate-rich plants (Rumex obtusifolius), possessed a unique mechanism for accumulating unusually high amounts of lactate. Similarly, larva of butterfly (Papilio machaon) fed with fennel (Foeniculum vulgare) accumulated lactate. Same butterfly also showed the elevated level of glycerol-3-phosphate equivalent to lactate. These evidences provide new insights into the mechanism underlying metabolite alteration between host plants and insect herbivores.     

Innate Immune Signaling Induces Interleukin-7 Production from Salivary Gland Cells and Accelerates the Development of Primary Sj?gren’s Syndrome in a Mouse Model

Elevated IL-7 in the target tissues is closely associated with multiple autoimmune disorders, including Sjögren’s syndrome (SS). We recently found that IL-7 plays an essential role in the development and onset of primary SS (pSS) in C57BL/6.NOD-Aec1Aec2 mice, a well-defined mouse model of primary SS. However, environmental signals that cause excessive IL-7 production are not well-characterized. Innate immune signaling plays a critical role in shaping the adaptive immune responses including autoimmune responses. We and others have previously shown that innate immune signaling can induce IL-7 expression in lungs and intestines of C57BL/6 mice. In this study, we characterized the effects of poly I:C, a double-stranded RNA analog and toll-like receptor 3 agonist, on the induction of IL-7 expression in salivary glands and on pSS development. We showed that poly I:C administration to C57BL/6 mice rapidly induced IL-7 expression in the salivary glands in a type 1 IFN- and IFN-γ-dependent manner. Moreover, poly I:C-induced IL-7 contributed to the optimal up-regulation of CXCL9 in the salivary glands, which may subsequently promote recruitment of more IFN-γ-producing T cells. Repeated administration of poly I:C to C57BL/6.NOD-Aec1Aec2 mice accelerated the development of SS-like exocrinopathy, and this effect was abolished by the blockade of IL-7 receptor signaling with a neutralizing antibody. Finally, poly I:C or a combination of IFN-α and IFN-γ induced IL-7 gene expression and protein production in a human salivary gland epithelial cell line. Hence, we demonstrate that IL-7 expression in the salivary gland cells can be induced by poly I:C and delineate a crucial mechanism by which innate immune signals facilitate the development of pSS, which is through induction of IL-7 in the target tissues.     

Phosphorus toxicity disrupts Rubisco activation and reactive oxygen species defence systems by phytic acid accumulation in leaves

Phosphorus (P) is an essential mineral nutrient for plants. Nevertheless, excessive P accumulation in leaf mesophyll cells causes necrotic symptoms in land plants; this phenomenon is termed P toxicity. However, the detailed mechanisms underlying P toxicity in plants have not yet been elucidated. This study aimed to investigate the molecular mechanism of P toxicity in rice. We found that under excessive inorganic P (Pi) application, Rubisco activation decreased and photosynthesis was inhibited, leading to lipid peroxidation. Although the defence systems against reactive oxygen species accumulation were activated under excessive Pi application conditions, the Cu/Zn-type superoxide dismutase activities were inhibited. A metabolic analysis revealed that excessive Pi application led to an increase in the cytosolic sugar phosphate concentration and the activation of phytic acid synthesis. These conditions induced mRNA expression of genes that are activated under metal-deficient conditions, although metals did accumulate. These results suggest that P toxicity is triggered by the attenuation of both photosynthesis and metal availability within cells mediated by phytic acid accumulation. Here, we discuss the whole phenomenon of P toxicity, beginning from the accumulation of Pi within cells to death in land plants.     

Distance-based analysis of variance for brain connectivity

The field of neuroimaging dedicated to mapping connections in the brain is increasingly being recognized as key for understanding neurodevelopment and pathology. Networks of these connections are quantitatively represented using complex structures, including matrices, functions, and graphs, which require specialized statistical techniques for estimation and inference about developmental and disorder-related changes. Unfortunately, classical statistical testing procedures are not well suited to high-dimensional testing problems. In the context of global or regional tests for differences in neuroimaging data, traditional analysis of variance (ANOVA) is not directly applicable without first summarizing the data into univariate or low-dimensional features, a process that might mask the salient features of high-dimensional distributions. In this work, we consider a general framework for two-sample testing of complex structures by studying generalized within-group and between-group variances based on distances between complex and potentially high-dimensional observations. We derive an asymptotic approximation to the null distribution of the ANOVA test statistic, and conduct simulation studies with scalar and graph outcomes to study finite sample properties of the test. Finally, we apply our test to our motivating study of structural connectivity in autism spectrum disorder.     

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L⁻¹·day⁻¹, exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.     

The SNP in the promoter region of the bovine ELOVL5 gene influences economic traits including subcutaneous fat thickness

Genetic analyses have contributed to improvements of economically important traits derived from adipose tissue such as fatty acid composition in beef. Elongation of very long chain fatty acids (ELOVL) genes encode for the enzymes that play an important role in elongation of long-chain fatty acids. In this study, we aimed to discover genetic polymorphisms of ELOVL gene family in cattle populations to develop genetic markers. As a result, five synonymous mutations were detected in the coding regions of the ELOVL1, ELOVL2, ELOVL3 and ELOVL5 genes. In addition, six mutations were identified in promoter region of the ELOVL5. Two of five mutations in the promoter region of ELOVL5 were expected to alter the ELOVL5 expression and influence the economic traits, because of the high synteny of the region which was essential for activation of Elovl5 in mouse. Therefore, we performed association analysis between the genotypes and traits and our result revealed that T allele of g.-110T>C in ELOVL5 gene promoter indicated significantly thinner subcutaneous fat thickness (TT, 2.39 cm; CT, 2.35 cm) than that of C allele (CC, 2.68 cm) in a Japanese Black population. Our results suggest that the g.-110T>C is a useful genetic marker for the breeding in beef cattle.     

Regional Differences in Elements of Human Peroneus LongusTendons

Many studies have been performed on the structure, molecular composition, and biochemical properties of tendons. However, comparatively little research has been conducted on the content of various trace elements within tendons. Six elements were analyzed in four regions of the peroneus longus tendon: the tensional part of the tendon immediately proximal to the lateral malleolus (region A), the compressive region of the tendon in contact with the lateral malleolus (region B), the compressive region of the tendon in contact with the deep surface of the cuboid (region C), and the tensional part of the tendon between the cuboid and first metatarsal, to which the tendon is attached (region D). Regions B and C are wraparound regions. The calcium content was higher in region C (2.10?±?0.93 mg/g) than in both regions A (1.25?±?0.51 mg/g) and D (1.43?±?0.41 mg/g) (p?<?0.05), indicating that it is likely related to regional differences in cartilage degeneration. The phosphorus content was also higher in region C, possibly because of low alkaline phosphatase activity in this region. The sulfur content was higher in the wraparound regions (region B: 0.98?±?0.09 mg/g, region C: 1.24?±?0.19 mg/g) than in both regions A (0.83?±?0.11 mg/g) and D (0.83?±?0.1 mg/g) (p?<?0.01); sulfur content is thought to be influenced by tendon–bone compression. Finally, the magnesium content in the wraparound regions was also higher, which is probably related to a higher level of fibrocartilage. No significant relationships were found with regard to zinc or iron. Overall, the findings of the present study indicate that element contents are related to function and anatomical differences in tendons, and that they may even vary within the same tendon.