Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase

Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H(2)O(2) exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris-HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35 min at 80 °C) and T(m) (from 73 to 77.5 °C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H(2)O(2) stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H(2)O(2) tolerance in concentrated buffer.     

Significant enhancement of lignan accumulation in hairy root cultures of Linum album using biotic elicitors

Linum album has been shown to accumulate some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, we examined the effects of fungal elicitors on the production of lignans in L. album hairy root cultures. The biosynthesis of lignans was differentially affected by fungal elicitors. Fusarium graminearum extract induced the highest increase of PTOX, 190 μg g^?1 dry weight (DW), and lariciresinol, 260 μg g^?1 DW, which was two-fold and three-fold greater than the untreated control, respectively, while Trichoderma viride extract enhanced the accumulation of MPTOX, instead of PTOX, up to 160 µg g^?1 DW, which was 2.4-fold greater than the control. The enhancing effects of fungal elicitors on lignans production was correlated with the increased expression of some key genes involved in the biosynthesis of these compounds, phenylalanine ammonia-lyase, cinnamoyl-CoA reductase, cinnamyl-alcohol dehydrogenase and pinoresinol-lariciresinol reductase.     

Polarization of Prostate Cancer-associated Macrophages Is Induced by Milk Fat Globule-EGF Factor 8 (MFG-E8)-mediated Efferocytosis

Tumor cells secrete factors that modulate macrophage activation and polarization into M2 type tumor-associated macrophages, which promote tumor growth, progression, and metastasis. The mechanisms that mediate this polarization are not clear. Macrophages are phagocytic cells that participate in the clearance of apoptotic cells, a process known as efferocytosis. Milk fat globule- EGF factor 8 (MFG-E8) is a bridge protein that facilitates efferocytosis and is associated with suppression of proinflammatory responses. This study investigated the hypothesis that MFG-E8-mediated efferocytosis promotes M2 polarization. Tissue and serum exosomes from prostate cancer patients presented higher levels of MFG-E8 compared with controls, a novel finding in human prostate cancer. Coculture of macrophages with apoptotic cancer cells increased efferocytosis, elevated MFG-E8 protein expression levels, and induced macrophage polarization into an alternatively activated M2 phenotype. Administration of antibody against MFG-E8 significantly attenuated the increase in M2 polarization. Inhibition of STAT3 phosphorylation using the inhibitor Stattic decreased efferocytosis and M2 macrophage polarization in vitro, with a correlating increase in SOCS3 protein expression. Moreover, MFG-E8 knockdown tumor cells cultured with wild-type or MFG-E8-deficient macrophages resulted in increased SOCS3 expression with decreased STAT3 activation. This suggests that SOCS3 and phospho-STAT3 act in an inversely dependent manner when stimulated by MFG-E8 and efferocytosis. These results uncover a unique role of efferocytosis via MFG-E8 as a mechanism for macrophage polarization into tumor-promoting M2 cells.     

Wild relatives of wheat: <Emphasis Type="Italic">Aegilops</Emphasis>–<Emphasis Type="Italic">Triticum</Emphasis> accessions disclose differential antioxidative and physiological responses to water stress

Wild relatives of wheat are an outstanding source of resistance to both abiotic and biotic stresses. In the present study, we evaluated the activity of four antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX)—along with photosynthetic pigments and shoot biomass in 12 Aegilops–Triticum accessions with different genomic constitutions and two tolerant and sensitive control varieties under well-watered (WW; 90% FC), moderate (MS; 50% FC) and severe (SS; 25% FC) water stress treatments. The analysis of variance for measured traits indicated highly significant effects of the water stress treatments, accessions, and their interactions. The 12 domesticated and wild relatives of wheat exhibited more variability and greater activity in the expression of antioxidative enzymes than cultivated wheats. While domesticated forms of wheat, T. aestivum (AABBDD) and T. durum (AABB) seem to have a functionally active antioxidant mechanism, other accessions with alien genomes—Ae. umbellulata (UU), Ae. crassa (MMDD), Ae. caudata (CC), Ae. cylindrica (DDCC) and T. boeoticum (A^bA^b)—respond to water stress by increasing enzymatic antioxidants as the dominant mechanism that contributes to the retention of oxidative balance in the cell. Furthermore, abovementioned accessions with alien genomes had higher photosynthetic pigment contents (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid) under water stress than well-watered conditions. Hence, these accessions could be used in future breeding programs to combine beneficial stress-adaptive characters of alien genomes into synthetic hexaploid wheat varieties in the field, even at limited water supply.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K_m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ～85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ～64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Network analysis of a proposed exit pathway for protons to the P-side of cytochrome c oxidase

Cytochrome c Oxidase (CcO) reduces O₂, the terminal electron acceptor, to water in the aerobic, respiratory electron transport chain. The energy released by O₂ reductions is stored by removing eight protons from the high pH, N-side, of the membrane with four used for chemistry in the active site and four pumped to the low pH, P-side. The proton transfers must occur along controllable proton pathways that prevent energy dissipating movement towards the N-side. The CcO N-side has well established D- and K-channels to deliver protons to the protein interior. The P-side has a buried core of hydrogen-bonded protonatable residues designated the Proton Loading Site cluster (PLS cluster) and many protonatable residues on the P-side surface, providing no obvious unique exit. Hydrogen bond pathways were identified in Molecular Dynamics (MD) trajectories of Rb. sphaeroides CcO prepared in the P_R state with the heme a₃ propionate and Glu286 in different protonation states. Grand Canonical Monte Carlo sampling of water locations, polar proton positions and residue protonation states in trajectory snapshots identify a limited number of water mediated, proton paths from PLS cluster to the surface via a (P-exit) cluster of residues. Key P-exit residues include His93, Ser168, Thr100 and Asn96. The hydrogen bonds between PLS cluster and P-exit clusters are mediated by a water wire in a cavity centered near Thr100, whose hydration can be interrupted by a hydrophobic pair, Leu255B (near Cu_A) and Ile99. Connections between the D channel and PLS via Glu286 are controlled by a second, variably hydrated cavity.