Directed evolution of a highly active Yersinia mollaretii phytase

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073?U/mg which is ～10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ～8,400 clones) a phytase variant with ～20% improved thermostability (58°C for 20?min; residual activity wild type ～34%; variant ～53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993?U/mg).     

Meniscofemoral ligaments--structural and material properties

The meniscofemoral ligaments (MFLs) of 28 human cadaveric knees were studied to determine their incidence, structural and material properties. Using the Race–Amis casting method for measurement, the mean cross-sectional area for the anterior MFL (aMFL) was 14.7 mm² (±14.8 mm²) whilst that of the posterior MFL (pMFL) was 20.9 mm² (±11.6 mm²). The ligaments were isolated and tensile tested in a materials testing machine. The mean loads to failure were 300.5 N (±155.0 N) for the aMFL and 302.5 N (±157.9 N) for the pMFL, with elastic moduli of 281 (±239 MPa) and 227 MPa (±128 MPa), respectively. These significant anatomical and material properties suggest a function for the MFL in the biomechanics of the knee, and should be borne in mind when considering hypotheses on MFL function. Such hypotheses include roles for the ligaments in knee stability and guiding meniscal motion.     

A unique FGF23 with the ability to activate FGFR signaling through both αKlotho and βKlotho

Three fibroblast growth factor (FGF) molecules, FGF19, FGF21, and FGF23, form a unique subfamily that functions as endocrine hormones. FGF19 and FGF21 can regulate glucose, lipid, and energy metabolism, while FGF23 regulates phosphate homeostasis. The FGF receptors and co-receptors for these three FGF molecules have been identified, and domains important for receptor interaction and specificity determination are beginning to be elucidated. However, a number of questions remain unanswered, such as the identification of fibroblast growth factor receptor responsible for glucose regulation. Here, we have generated a variant of FGF23: FGF23-21c, where the C-terminal domain of FGF23 was replaced with the corresponding regions from FGF21. FGF23-21c showed a number of interesting and unexpected properties in vitro. In contrast to wild-type FGF23, FGF23-21c gained the ability to activate FGFR1c and FGFR2c in the presence of βKlotho and was able to stimulate glucose uptake into adipocytes in vitro and lower glucose levels in ob/ob diabetic mice model to similar extent as FGF21 in vivo. These results suggest that βKlotho/FGFR1c or FGFR2c receptor complexes are sufficient for glucose regulation. Interestingly, without the FGF23 C-terminal domain, FGF23-21c was still able to activate fibroblast growth factor receptors in the presence of αKlotho. This suggests not only that sequences outside of the C-terminal region may also contribute to the interaction with co-receptors but also that FGF23-21c may be able to regulate both glucose and phosphate metabolisms. This raises an interesting concept of designing an FGF molecule that may be able to address multiple diseases simultaneously. Further understanding of FGF/receptor interactions may allow the development of exciting opportunities for novel therapeutic discovery.     

Effect of different cultivation conditions and inducers on the production of laccase by the litter-dwelling fungal isolate Fusarium incarnatum LD-3 under solid substrate fermentation

The litter-dwelling fungus Fusarium incarnatum LD-3 has been identified as a novel producer of laccase. The present work was oriented towards the optimization of various cultivation conditions for maximizing laccase production under solid substrate fermentation. The process parameters were optimized by the “one factor at a time” approach. Maximum laccsase production was obtained at pH 5.0 and at a temperature of 28 °C with 60 % moisture content using rice bran as a substrate. The laccase production was enhanced in the presence of aromatic inducer, i.e. ortho-dianisidine at a concentration of 0.5 mM. Laccase production was further increased by 52.56 % when the medium was supplemented with 2 % (v/v) alcohol. Among the various amino acids tested as a growth factor and nitrogen source, D-Serine and DL-2 Amino n-butyric acid, DL-Alanine and L-Glycine were found to be the most suitable for laccase production. The highest laccase production (1,352.64 U/g) was achieved under optimized conditions, and was 2.1-fold higher than the unoptimized conditions. Thus, the novel litter-dwelling fungal isolate Fusarium incarnatum LD-3 seems to be an efficient producer of laccase and can be further exploited for biotechnological applications. This is the first report on the optimization of cultivation conditions and inducers for laccase production from Fusarium incarnatum LD-3.     

Design and synthesis of MMP inhibitors with appended fluorescent tags for imaging and visualization of matrix metalloproteinase enzymes

We describe the synthesis, MMP-2 and 9 potency, and in vitro evaluation of a series of α-sulfone hydroxmate MMP inhibitors conjugated to a series of dyes with different absorption/emission lamina maxima’s that can be used to visualize tumors.     

Galectin-8 Ameliorates Murine Autoimmune Ocular Pathology and Promotes a Regulatory T Cell Response

Galectins have emerged as potent immunoregulatory agents that control chronic inflammation through distinct mechanisms. Here, we report that treatment with Galectin-8 (Gal-8), a tandem-repeat member of the galectin family, reduces retinal pathology and prevents photoreceptor cell damage in a murine model of experimental autoimmune uveitis. Gal-8 treatment increased the number of regulatory T cells (Treg) in both the draining lymph node (dLN) and the inflamed retina. Moreover, a greater percentage of Treg cells in the dLN and retina of Gal-8 treated animals expressed the inhibitory coreceptor cytotoxic T lymphocyte antigen (CTLA)-4, the immunosuppressive cytokine IL-10, and the tissue-homing integrin CD103. Treg cells in the retina of Gal-8-treated mice were primarily inducible Treg cells that lack the expression of neuropilin-1. In addition, Gal-8 treatment blunted production of inflammatory cytokines by retinal T helper type (T_H) 1 and T_H17 cells. The effect of Gal-8 on T cell differentiation and/or function was specific for tissues undergoing an active immune response, as Gal-8 treatment had no effect on T cell populations in the spleen. Given the need for rational therapies for managing human uveitis, Gal-8 emerges as an attractive therapeutic candidate not only for treating retinal autoimmune diseases, but also for other T_H1- and T_H17-mediated inflammatory disorders.     

Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH

Vascular smooth muscle (VSM) derived from pulmonary arteries generally contract to hypoxia, whereas VSM from systemic arteries usually relax, indicating the presence of basic oxygen-sensing mechanisms in VSM that are adapted to the environment from which they are derived. This review considers how fundamental processes associated with the generation of reactive oxygen species (ROS) by oxidase enzymes, the metabolic control of cytosolic NADH, NADPH and glutathione redox systems, and mitochondrial function interact with signaling systems regulating vascular force in a manner that is potentially adapted to be involved in Po2 sensing. Evidence for opposing hypotheses of hypoxia, either decreasing or increasing mitochondrial ROS, is considered together with the Po2 dependence of ROS production by Nox oxidases as sensors potentially contributing to hypoxic pulmonary vasoconstriction. Processes through which ROS and NAD(P)H redox changes potentially control interactive signaling systems, including soluble guanylate cyclase, potassium channels, and intracellular calcium are discussed together with the data supporting their regulation by redox in responses to hypoxia. Evidence for hypothesized potential differences between systemic and pulmonary arteries originating from properties of mitochondrial ROS generation and the redox sensitivity of potassium channels is compared with a new hypothesis in which differences in the control of cytosolic NADPH redox by the pentose phosphate pathway results in increased NADPH and Nox oxidase-derived ROS in pulmonary arteries, whereas lower levels of glucose-6-phosphate dehydrogenase in coronary arteries may permit hypoxia to activate a vasodilator mechanism controlled by oxidation of cytosolic NADPH.     

Activation of Glucose-6-phosphate Dehydrogenase Promotes Acute Hypoxic Pulmonary Artery Contraction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O₂ tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N₂, 5% CO₂) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD⁺ and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca²⁺ sensitivity to the myofilaments and diminished Ca²⁺-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca²⁺-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD^mut(−/−) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP⁺ ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.     

Elucidation of signaling and functional activities of an orphan GPCR, GPR81

GPR81 is an orphan G protein-coupled receptor (GPCR) that has a high degree of homology to the nicotinic acid receptor GPR109A. GPR81 expression is highly enriched and specific in adipocytes. However, the function and signaling properties of GPR81 are unknown because of the lack of natural or synthetic ligands. Using chimeric G proteins that convert Gi-coupled receptors to Gq-mediated inositol phosphate (IP) accumulation, we show that GPR81 can constitutively increase IP accumulation in HEK293 cells and suggest that GPR81 couples to the Gi signaling pathway. We also constructed a chimeric receptor that expresses the extracellular domains of cysteinyl leukotriene 2 receptor (CysLT2R) and the intracellular domains of GPR81. We show that the CysLT2R ligand, leukotriene D(4) (LTD4), is able to activate this chimeric receptor through activation of the Gi pathway. In addition, LTD4 is able to inhibit lipolysis in adipocytes expressing this chimeric receptor. These results suggest that GPR81 couples to the Gi signaling pathway and that activation of the receptor may regulate adipocyte function and metabolism. Hence, targeting GPR81 may lead to the development of a novel and effective therapy for dyslipidemia and a better side effect profile than nicotinic acid.