Shift to Pseudomonic acid B production in P. fluorescens NCIMB10586 by mutation of mupirocin tailoring genes mupO, mupU, mupV, and macpE

Mupirocin, a polyketide-derived antibiotic from Pseudomonas fluorescens NCIMB10586, is a mixture of pseudomonic acids (PA) that target isoleucyl-tRNA synthase. The mup gene cluster encodes both type I polyketide synthases and monofunctional enzymes that should play a role during the conversion of the product of the polyketide synthase into the active antibiotic (tailoring). By in-frame deletion analysis of selected tailoring open-reading frames we show that mupQ, mupS, mupT, and mupW are essential for mupirocin production, whereas mupO, mupU, mupV, and macpE are essential for production of PA-A but not PA-B. Therefore, PA-B is not simply produced by hydroxylation of PA-A but is either a precursor of PA-A or a shunt product. In the mupW mutant, a new metabolite lacking the tetrahydropyran ring is produced, implicating mupW in oxidation of the 16-methyl group.     

Fe2+ and Cu2+ Increase the Production of Hyaluronic Acid by Lactobacilli via Affecting Different Stages of the Pentose Phosphate Pathway

This study aimed at optimizing the production of hyaluronic acid by Lactobacillus acidophilus FTDC 1231 using response surface methodology and evaluating the effects of divalent metal ions along the production pathway using molecular docking. Among different divalent metal ions that were screened, only iron (II) sulphate and copper (II) sulphate significantly (P?<?0.05) affected the production of hyaluronic acid. Subsequent optimization yielded hyaluronic acid at concentration of 0.6152?mg/mL in the presence of 1.24 mol L^?1 iron (II) sulphate and 0.16 mol L^?1 of copper (II) sulphate (103 % increase compared to absence of divalent metal ions). Data from molecular docking showed Fe²⁺ improved the binding affinity of UDP-pyrophophorylase towards glucose-1-phosphate, while Cu²⁺ contributed towards the interaction between UDP-glucose dehydrogenase and UDP-glucose. We have demonstrated that lactobacilli could produce hyaluronic acid at increased concentration upon facilitation by specific divalent metal ions, via specific targets of enzymes and substrates along pentose phosphate pathway.     

Methods for Studying Endocytotic Pathways of Herpesvirus Encoded G Protein-Coupled Receptors

Endocytosis is a fundamental process involved in trafficking of various extracellular and transmembrane molecules from the cell surface to its interior. This enables cells to communicate and respond to external environments, maintain cellular homeostasis, and transduce signals. G protein-coupled receptors (GPCRs) constitute a family of receptors with seven transmembrane alpha-helical domains (7TM receptors) expressed at the cell surface, where they regulate physiological and pathological cellular processes. Several herpesviruses encode receptors (vGPCRs) which benefits the virus by avoiding host immune surveillance, supporting viral dissemination, and thereby establishing widespread and lifelong infection, processes where receptor signaling and/or endocytosis seem central. vGPCRs are rising as potential drug targets as exemplified by the cytomegalovirus-encoded receptor US28, where its constitutive internalization has been exploited for selective drug delivery in virus infected cells. Therefore, studying GPCR trafficking is of great importance. This review provides an overview of the current knowledge of endocytic and cell localization properties of vGPCRs and methodological approaches used for studying receptor internalization. Using such novel approaches, we show constitutive internalization of the BILF1 receptor from human and porcine γ-1 herpesviruses and present motifs from the eukaryotic linear motif (ELM) resources with importance for vGPCR endocytosis.     

Effects of ultrasound on growth, bioconversion of isoflavones and probiotic properties of parent and subsequent passages of Lactobacillus fermentum BT 8633 in biotin-supplemented soymilk

This study aimed to evaluate the effects of ultrasound on Lactobacillus fermentum BT 8633 in parent and subsequent passages based on their growth and isoflavone bioconversion activities in biotin-supplemented soymilk. The treated cells were also assessed for impact of ultrasound on probiotic properties. The growth of ultrasonicated parent cells increased (P < 0.05) by 3.23-9.14% compared to that of the control during fermentation in biotin-soymilk. This was also associated with enhanced intracellular and extracellular (8.4-17.0% and 16.7-49.2%, respectively; P < 0.05) β-glucosidase specific activity, leading to increased bioconversion of isoflavones glucosides to aglycones during fermentation in biotin-soymilk compared to that of the control (P < 0.05). Such traits may be credited to the reversible permeabilized membrane of ultrasonicated parent cells that have facilitated the transport of molecules across the membrane. The growing characteristics of first, second and third passage of treated cells in biotin-soymilk were similar (P > 0.05) to that of the control, where their growth, enzyme and isoflavone bioconversion activities (P > 0.05) were comparable. This may be attributed to the temporary permeabilization in the membrane of treated cells. Ultrasound affected probiotic properties of parent L. fermentum, by reducing tolerance ability towards acid (pH 2) and bile; lowering inhibitory activities against selected pathogens and reducing adhesion ability compared to that of the control (P < 0.05). The first, second and third passage of treated cells did not exhibit such traits, with the exception of their bile tolerance ability which was inherited to the first passage (P < 0.05). Our results suggested that ultrasound could be used to increase bioactivity of biotin-soymilk via fermentation by probiotic L. fermentum FTDC 8633 for the development of functional food.     

1H‐Pyrrole‐2‐carbothioamide

The reaction of ethoxy­carbonyl­pyrrole‐2‐thio­carbox­amide with sodium hydro­xide affords the known title compound, C₅H₆N₂S, whose structure shows a three‐dimensional hydrogen‐bonded lattice.     

Construction of a pH-driven supramolecular nanovalve

[Structure: see text] The versatility of supramolecular chemistry has been exploited in constructing nanovalves based on mesoporous silica MCM-41 and the mutual recognition between secondary dialkylammonium ions and dibenzo[24]crown-8 (DB24C8). Naphthalene-containing dialkylammonium threads were tethered to the MCM-41, followed by loading with coumarin 460 and capping with DB24C8. Controlled release of coumarin 460 from the pores of MCM-41 was demonstrated using different bases. The rate of release of coumarin 460 from the nanovalves depends on the size of the base.     

Implementation of a cohesive zone model for the investigation of the dynamic behavior of a rotating shaft with a transverse crack

The influence of a transverse crack on the vibration of a rotating shaft has been at the focus of attention of many researchers. The knowledge of the dynamic behavior of cracked shaft has helped in predicting the presence of a crack in a rotor. Here, the changing stiffness of the cracked shaft is investigated based on a cohesive zone model. This model is developed for mode-I plane strain and accounts for triaxiality of the stress state explicitly by using basic elastic-plastic constitutive relations. Then, the proposed numerical solution is compared to the switching crack model, which is based on linear elastic fracture mechanics. The cohesive zone model is implemented in finite element techniques to predict and to analyse the dynamic behavior of cracked rotor system. Timoshenko beam theory is used to model the discrete shaft under the effect of gravity, unbalance force and gyroscopic effect. The analysis includes the cohesive function for describing the breathing crack and the reduction of the second moment of area of the element at the location of the crack. (© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)     

Polarizability anisotropies of cyano-substituted azulene,biphenyl, 2,2'- bipyridyl and naphthalene

The conformations of a number of cyano-substituted aromatic model mesogens with actual or potential application in liquid crystals have been obtained using density functional B-LYP/6-311G(d) methodology. Dipole polarizability tensors have been computed at Hartree-Fock and B3-LYP levels of theory using Spackman's recommended basis sets, in order to obtain polarizability anisotropies, and to compare them with those of the parent compounds. The results indicate that 6-cyanoazulene is a potentially important mesogenic component, having a polarizability anisotropy not much less than 4-cyano-2,2'-bipyridyl. Electric dipole transition moments and oscillator strengths to the lowest singlet and triplet excited states have also been estimated for the cyanoazulenes, using a Configuration-Interaction Singles CIS/6-311G(d) approximation.     

Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586

The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus. The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified. The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS). Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects. A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.