The access to the binding cavity of Lipocalin-type Prostaglandin D-synthase (L-PGDS) is regulated by a Tryptophan gate

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2 (prostaglandin H2) to produce PGD2 (prostaglandin D2) with 9-hydroxy and 11-keto groups. The product of the reaction, PGD2, is the precursor of several metabolites involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as -trace protein, the second most abundant protein in human cerebrospinal fluid. Previous structural work on the mouse and human proteins has focused on the identification of the amino acids responsible and the proposal of a mechanism for catalysis. In this paper, the X-ray structures of the apo and holo forms (bound to PEG) of the C65A mutant of human L-PGDS at 1.40 A \u2da resolution and of the double mutant C65A/K59A at 1.60 A \u2da resolution are reported. The apo forms of the double mutants C65A/W54F and C65A/W112F and the triple mutant C65A/W54F/W112F have also been studied. Mutation of the lysine residue does not seem to affect the binding of PEG to the ligand-binding cavity, and mutation of a single or both tryptophans appears to have the same effect on the position of these two aromatic residues at the entrance to the cavity. A solvent molecule has also been identified in an invariant position in the cavity of virtually all of the molecules present in the nine asymmetric units of the crystals that have been examined. Taken together, these observations indicate that the residues that have been mutated indeed appear to play a role in the entrance\u2013exit process of the substrate and/or other ligands into/out of the binding cavity of the lipocalin

Overexpression of a Bacillus subilis amylase in E.coli and application in bread making

Bacterial alpha-amylases (EC3.2.1.1) have potential use in wide number of industrial applications such as textile, paper, detergent, food, fermentation and pharmaceutical industries. Recombinant DNA technology for amylase production involves the selection of an amylase gene, its insertion into an appropriate vector system, transformation in an efficient bacterial system to produce high amount of recombinant protein. In this context, the aim of this work is the overexpression of an α-amylase gene from Bacillus subtilis US572 in E.coli strain, the characterization of the recombinant enzyme and test the effect of different quantities added of amylase on wheat flours and bread characterization

Structure/function/properties relationships and application of a GH11 xylanase

Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of the heteroxylans constituting the lignocellulosic plant cell wall. Due to their variety, xylanases have been classified in glycoside hydrolase families GH5, GH8, GH10, GH11, GH30 and GH43 in the CAZy database. In this work, we focus on GH11 family, which is one of the best characterized GH families with bacterial and fungal members. GH11 xylanases have for a long time been used as biotechnological tools in various industrial applications and represent in addition promising candidates for future other uses

Oxyresveratrol-Loaded PLGA Nanoparticles Inhibit Oxygen Free Radical Production by Human Monocytes: Role in Nanoparticle Biocompatibility

Oxyresveratrol, a polyphenol extracted from the plant Artocarpus lakoocha Roxb, has been reported to be an antioxidant and an oxygen-free radical scavenger. We investigated whether oxyresveratrol affects the generation of superoxide anion (O2 ) by human monocytes, which are powerful reactive oxygen species (ROS) producers. We found that oxyresveratrol inhibited the O2 production induced upon stimulation of monocytes with -glucan, a well known fungal immune cell activator. We then investigated whether the inclusion of oxyresveratrol into nanoparticles could modulate its effects on O2 release. We synthesized poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and we assessed their effects on monocytes. We found that empty PLGA nanoparticles induced O2 production by resting monocytes and enhanced the formation of this radical in -glucan-stimulated monocytes. Interestingly, the insertion of oxyresveratrol into PLGA nanoparticles significantly inhibited the O2 production elicited by unloaded nanoparticles in resting monocytes as well as the synergistic effect of nanoparticles and -glucan. Our results indicate that oxyresveratrol is able to inhibit ROS production by activated monocytes, and its inclusion into PLGA nanoparticles mitigates the oxidative effects due to the interaction between these nanoparticles and resting monocytes. Moreover, oxyresveratrol can contrast the synergistic effects of nanoparticles with fungal agents that could be present in the patient tissues. Therefore, oxyresveratrol is a natural compound able to make PLGA nanoparticles more biocompatible

Oxyresveratrol Inhibits R848-Induced Pro-Inflammatory Mediators Release by Human Dendritic Cells Even When Embedded in PLGA Nanoparticles

Oxyresveratrol, a stilbene extracted from the plant Artocarpus lakoocha Roxb., has been reported to provide a considerable anti-inflammatory activity. Since the mechanisms of this therapeutic action have been poorly clarified, we investigated whether oxyresveratrol affects the release of the pro-inflammatory cytokines IL-12, IL-6, and TNF-\u3b1 by human dendritic cells (DCs). We found that oxyresveratrol did not elicit per se the release of these cytokines, but inhibited their secretion induced upon DC stimulation with R848 (Resiquimod), a well-known immune cell activator en-gaging receptors recognizing RNA viruses. We then investigated whether the inclusion of ox-yresveratrol into nanoparticles promoting its ingestion by DCs could favor its effects on cytokine release. For this purpose we synthesized and characterized poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and we assessed their effects on DCs. We found that bare PLGA nanoparticles did not affect cytokine secretion by resting DCs, but increased IL-12, IL-6, and TNF-\u3b1 secretion by R848-stimulated DCs, an event known as \u201cpriming effect\u201d. We then loaded PLGA nanoparticles with oxyresveratrol and we observed that oxyresveratrol-bearing particles did not stimulate the cytokine release by resting DCs and inhibited the PLGA-dependent enhancement of IL-12, IL-6, and TNF-\u3b1 secretion by R848-stimulated DCs. The results herein reported indicate that oxyresveratrol suppresses the cytokine production by activated DCs, thus representing a good anti-inflammatory and immune-suppressive agent. Moreover, its inclusion into PLGA nanoparticles mitigates the pro-inflammatory effects due to cooperation between nanoparticles and R848 in cytokine release. Therefore, oxyresveratrol can be able to contrast the synergistic effects of nanoparticles with microorganisms that could be present in the patient tissues, therefore overcoming a condition unfavorable to the use of some nanoparticles in biological systems

High resolution crystal structure data of human plasma retinol-binding protein (RBP4) bound to retinol and fatty acids

Retinol is transported in vertebrate plasma bound to a protein called retinol-binding protein (RBP4) so far believed to be specific for the vitamin. When the protein is saturated with retinol it binds tightly to another plasma protein, transthyretin while when not saturated with retinol it does not bind to TTR (Goodman, 1984). The X-ray structures of human RBP4, holo and devoid of retinol in its binding site are known to resolutions of 2.0 and 2.5 \uc5 (Cowan et al., 1990; Zanotti et al., 1993) [2,3]. We have shown that RBP4 is not specific for retinol but it is also found in plasma, urine and amniotic fluid bound to fatty acids. Here we present 1.5 \uc5 resolution crystal data on human plasma retinol-binding protein bound to retinol and fatty acids. These are the highest resolution data available in the Protein Data Bank for this protein. For further details and experimental findings please refer to the article \u201c Human plasma retinol-binding protein (RBP4) is also a fatty acid-binding protein\u201d (Perduca et al., 2018) [4]

Ln(III) Complexes Embedded in Biocompatible PLGA Nanoparticles as Potential Vis-to-NIR Optical Probes

In this contribution, we present the spectroscopic study of two NIR emitting hydrophobic heteroleptic (R,R)-YbL1(tta) and (R,R)-NdL1(tta) complexes (with tta = 2-thenoyltrifluoroacetonate and L1 = N,N0 -bis(2-(8-hydroxyquinolinate)methylidene)-1,2-(R,R or S,S)-cyclohexanediamine), both in methanol solution and embedded in water dispersible and biocompatible poly lactic-co-glycolic acid (PLGA) nanoparticles. Thanks to their absorption properties in a wide range of wavelengths extending from the UV up to the blue and green visible regions, the emission of these complexes can be effectively sensitized using visible radiation, which is much less harmful to tissues and skin than the UV one. The encapsulation of the two Ln(III)-based complexes in PLGA allows us to preserve their nature, making them stable in water and to test their cytotoxicity on two different cell lines, with the aim of using them in the future as potential bioimaging optical probes

Two novel C-terminus RUNX2 mutations in two cleidocranial dysplasia (CCD) patients impairing p53 expression

Cleidocranial dysplasia (CCD), a dominantly inherited skeletal disease, is characterized by a variable phenotype ranging from dental alterations to severe skeletal defects. Either de novo or inherited mutations in the RUNX2 gene have been identified in most CCD patients. Transcription factor RUNX2, the osteogenic master gene, plays a central role in the commitment of mesenchymal stem cells to osteoblast lineage. With the aim to analyse the effects of RUNX2 mutations in CCD patients, we investigated RUNX2 gene expression and the osteogenic potential of two CCD patients’ cells. In addition, with the aim to better understand how RUNX2 mutations interfere with osteogenic differentiation, we performed string analyses to identify proteins interacting with RUNX2 and analysed p53 expression levels. Our findings demonstrated for the first time that, in addition to the alteration of downstream gene expression, RUNX2 mutations impair p53 expression affecting osteogenic maturation. In conclusion, the present work provides new insights into the role of RUNX2 mutations in CCD patients and suggests that an in-depth analysis of the RUNX2-associated gene network may contribute to better understand the complex molecular and phenotypic alterations in mutant subjects

Near Infared Circularly Polarized Luminescence from water stable organic nanoparticles containing a chiral Yb(III) complex

We report the first example of very efficient NIR Circularly Polarized Luminescence (CPL) (around 970 nm) in water, obtained thanks to the combined use of a chiral Yb complex and of poly lactic-co-glycolic acid (PLGA) nanoparticles. [Yb L (tta) 2 ]CH 3 COO ( L = N, N'-bis(2-pyridylmethylidene)-1,2-( R,R + S,S ) cyclohexanediamine and tta = 2-thenoyltrifluoroacetonate) shows good CPL in organic solvents, because the tta ligands efficiently sensitize Yb NIR luminescence and the readily prepared chiral ligand L endows the complex with the necessary dissymmetry. PLGA nanoparticles incorporate the complex and protect the metal ion from the intrusion of solvent molecules, while ensuring biocompatibility, water solubility and stability to the complex. Hydrophilic NIR-CPL optical probes can find applications in the field of NIR-CPL bio-assays

Structure and Properties of the C-terminal Domain of Insulin-like Growth Factor-binding Protein-1 Isolated from Human Amniotic Fluid

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules