Activation of Fly Ash–Lime Reactions: Kinetic Approach

Lime–fly ash reactions play a key role in improving the mechanical strength and tailoring the permeability characteristics of compacted fly ash. Activation of fly ash–lime pozzolanic reactions should accelerate the rate of strength development and possibly mobilize higher compressive strengths, facilitating improved engineering performance of fly ash amended materials. This paper makes an assessment of activation of lime–fly ash reactions by curing compacted fly ash–lime specimens at ambient (25°C) and at elevated temperature (80°C). The kinetics of fly ash–lime reactions are examined by monitoring the reacted lime as a function of curing period and temperature. The influence of variations in fly ash/lime content and dry density on the compressive strength developed by specimens at both temperatures is evaluated. The thermodynamic parameters for the fly ash–lime reactions have also been examined. Experimental results showed that curing at 80°C for 24 h accelerated fly ash–lime reactions such that it caused the steam cured (SC) specimens to evelop 1.21–2.44 fold larger strengths than room-temperature cured (RTC) specimens cured at 25°C for 28 days. Analysis of thermodynamic parameters indicated that the fly ash–lime reactions are thermodynamically favored at fly ash contents of 50–70% and lime additions of 16–20%, and the reactions are endothermic in nature. DOI: 10.1061/(ASCE)MT.1943-5533.0000482. © 2012 American Society of Civil Engineers.

Trm1p, a Zn(II)(2)Cys(6)-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris

The zinc finger transcription factors Mxr1p and Rop are key regulators of methanol metabolism in the methylotrophic yeast, Pichia pastoris, while Trm1p and Trm2p regulate methanol metabolism in Candida boidinii. Here, we demonstrate that Trm1p is essential for the expression of genes of methanol utilization (mut) pathway in P. pastoris as well. Expression of AOXI and other genes of mut pathway is severely compromised in P. pastoris Delta Trm1 strain resulting in impaired growth on media containing methanol as the sole source of carbon. Trm1p localizes to the nucleus of cells cultured on glucose or methanol. The zinc finger domain of Mxr1p but not Trm1p binds to AOXI promoter sequences in vitro, indicating that these two positive regulators act by different mechanisms. We conclude that both Trm1p and Mxr1p are essential for the expression of genes of mut pathway in P. pastoris and the mechanism of transcriptional regulation of mut pathway may be similar in P. pastoris and C. boidinii. (C) 2014 Elsevier Inc. All rights reserved.

Synthesis, characterization and reactivity studies of electrophilic ruthenium(II) complexes: a study of H-2 activation and labilization

Reaction of 2,2'-bipyridine (bpy) with dinuclear complexesRuCl(dfppe)(mu-Cl)(3)Ru(dmso-S)(3)](dfppe = 1,2-bis(dipentafluorophenyl phosphino)ethane (C6F5)(2)PCH2CH2P(C6F5)(2); dmso = dimethyl sulfoxide) (1) or RuCl(dfppe)(mu-Cl)(3)RuCl(dfppe)] (2) affords the mononuclear species trans-RuCl2(bpy)(dfppe)] (3). Using this precursor complex (3), a series of new cationic Ru(II) electrophilic complexes RuCl(L)(bpy)(dfppe)]Z] (L = P(OMe)(3) (5), PMe3 (6), CH3CN (7), CO (8), H2O (9); Z = OTf (5, 6, 7, 8), BAr4F (9) have been synthesized via abstraction of chloride by AgOTf or NaBAr4F in the presence of L. Complexes 5 and 6 were converted into the corresponding isomeric hydride derivatives RuH(PMe3)(bpy)(dfppe)]OTf] (10a, 10b) and RuH(P(OMe)(3))(bpy)(dfppe)]OTf] (11a, 11b) respectively, when treated with NaBH4. Protonation of the cationic monohydride complex (11a) with HOTf at low temperatures resulted in H-2 evolution accompanied by the formation of either solvent or triflate bound six coordinated species Ru(S)(P(OMe)(3))(bpy)(dfppe)]OTf](n) (S = solvent (n = 2), triflate (n = 1)] (13a/13b); these species have not been isolated and could not be established with certainty. They (13a/13b) were not isolated, instead the six-coordinated isomeric aqua complexes cis-(Ru(bpy)(dfppe)(OH2)(P(OMe)(3))]OTf](2) (14a/14b) were isolated. Reaction of the aqua complexes (14a/14b) with 1 atm of H-2 at room temperature in acetone-d(6) solvent resulted in heterolytic cleavage of the H-H bond. Results of the studies on H-2 lability and heterolytic activation using these complexes are discussed. The complexes 3, 5, 11a, and 14a have been structurally characterized.

Dual enzyme responsive and targeted nanocapsules for intracellular delivery of anticancer agents

We report the fabrication of dual enzyme responsive hollow nanocapsules which can be targeted to deliver anticancer agents specifically inside cancer cells. The enzyme responsive elements, integrated in the nanocapsule walls, undergo degradation in the presence of either trypsin or hyaluronidase leading to the release of encapsulated drug molecules. These nanocapsules, which were crosslinked and functionalised with folic acid, showed minimal drug leakage when kept in pH 7.4 PBS buffer, but released the drug molecules at a rapid rate in the presence of either one of the triggering enzymes. Studies on cellular interactions of these nanocapsules revealed that doxorubicin loaded nanocapsules were taken up by cervical cancer cells via folic acid receptor medicated endocytosis. Interestingly the nanocapsules were able to disintegrate inside the cancer cells and release doxorubicin which then migrated into the nucleus to induce cell death. This study indicates that these nanocapsules fabricated from biopolymers can serve as an excellent platform for targeted intracellular drug delivery to cancer cells.

Dual role of MsRbpA: transcription activation and rescue of transcription from the inhibitory effect of rifampicin

MsRbpA is an RNA polymerase (RNAP) binding protein from Mycobacterium smegmatis. According to previous studies, MsRbpA rescues rifampicin-induced transcription inhibition upon binding to the RNAP. Others have shown that RbpA from Mycobacterium tuberculosis (MtbRbpA) is a transcription activator. In this study, we report that both MsRbpA and MtbRbpA activate transcription as well as rescue rifampicin-induced transcription inhibition. Transcription activation is achieved through the increased formation of closed RNAP-promoter complex as well as enhanced rate of conversion of this complex to a stable transcriptionally competent RNAP promoter complex. When a 16 aa peptide fragment (Asp 58 to Lys 73) was deleted from MsRbpA, the resulting protein showed 1000-fold reduced binding with core RNAP. The deletion results in abolition of transcription activation and rescue of transcription from the inhibitory effect of rifampicin. Through alanine scanning of this essential region of MsRbpA, Gly 67, Val 69, Pro 70 and Pro 72 residues are identified to be important for MsRbpA function. Furthermore, we report here that the protein is indispensable for M. smegmatis, and it appears to help the organism grow in the presence of the antibiotic rifampicin.

Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis

We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.

Charge density distribution and electrostatic interactions of ethionamide: an inhibitor of the enoyl acyl carrier protein reductase (inhA) enzyme of Mycobacterium tuberculosis

An experimental charge density analysis of an anti-TB drug ethionamide was carried out from high resolution X-ray diffraction at 100 K to understand its charge density distribution and electrostatic properties. The experimental results were validated from periodic theoretical charge density calculations performed using CRYSTAL09 at the B3LYP/6-31G** level of theory. The electron density rho(bcp)(r) and the Laplacian of electron density del(2)(rho bcp)(r) of the molecule calculated from both the methods display the charge density distribution of the ethionamide molecule in the crystal field. The electrostatic potential map shows a large electropositive region around the pyridine ring and a large electronegative region at the vicinity of the thiol atom. The calculated experimental dipole moment is 10.6D, which is higher than the value calculated from theory (8.2D). The topological properties of C-H center dot center dot center dot S, N-H center dot center dot center dot N and N-H center dot center dot center dot S hydrogen bonds were calculated, revealing their strength. The charge density analysis of the ethionamide molecule determined from both the experiment and theory gives the topological and electrostatic properties of the molecule, which allows to precisely understand the nature of intra and intermolecular interactions.

Lactoylglutathione lyase, a critical enzyme in methylglyoxal detoxification, contributes to survival of Salmonella in the nutrient rich environment

Glyoxalase I which is synonymously known as lactoylglutathione lyase is a critical enzyme in methylglyoxal (MG) detoxification. We assessed the STM3117 encoded lactoylglutathione lyase (Lgl) of Salmonella Typhimurium, which is known to function as a virulence factor, due in part to its ability to detoxify methylglyoxal. We found that STM3117 encoded Lgl isomerises the hemithioacetal adduct of MG and glutathione (GSH) into S-lactoylglutathione. Lgl was observed to be an outer membrane bound protein with maximum expression at the exponential growth phase. The deletion mutant of S. Typhimurium (lgl) exhibited a notable growth inhibition coupled with oxidative DNA damage and membrane disruptions, in accordance with the growth arrest phenomenon associated with typical glyoxalase I deletion. However, growth in glucose minimal medium did not result in any inhibition. Endogenous expression of recombinant Lgl in serovar Typhi led to an increased resistance and growth in presence of external MG. Being a metalloprotein, Lgl was found to get activated maximally by Co2+ ion followed by Ni2+, while Zn2+ did not activate the enzyme and this could be attributed to the geometry of the particular protein-metal complex attained in the catalytically active state. Our results offer an insight on the pivotal role of the virulence associated and horizontally acquired STM3117 gene in non-typhoidal serovars with direct correlation of its activity in lending survival advantage to Salmonella spp.

Activation of InsP(3) receptors is sufficient for inducing graded intrinsic plasticity in rat hippocampal pyramidal neurons

The synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signaling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. In this study, we complemented the recently established necessity of inositol trisphosphate (InsP(3)) receptors (InsP(3)R) in a form of intrinsic plasticity by asking if InsP(3)R activation was sufficient to induce intrinsic plasticity in hippocampal neurons. Specifically, incorporation of D-myo-InsP(3) in the recording pipette reduced input resistance, maximal impedance amplitude, and temporal summation but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead. Strikingly, the magnitude of plasticity in all these measurements was dependent on InsP 3 concentration, emphasizing the graded dependence of such plasticity on InsP(3)R activation. Mechanistically, we found that this InsP(3)-induced plasticity depended on hyperpolarization-activated cyclic nucleotide-gated channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP(3)Rs, the influx of calcium through N-methyl-D-aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. Our results delineate a causal role for InsP(3)Rs in graded adaptation of neuronal response dynamics, revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis.

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-beta

Actions of transforming growth factor-beta are largely context dependent. For instance, TGF-beta is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-beta also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-beta would change in the context of transformation, we have compared the differential gene regulation by TGF-beta in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-beta in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-beta is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-beta. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-beta in hFhTERT cells and TGF-beta conferred action of these cells was mediated by WNT4. While TGF-beta activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-beta which matched with the response of HT1080 cells to TGF-beta. These data suggest context dependent activation of non-canonical signaling by TGF-beta. (C) 2015 Published by Elsevier Inc.

Remarkable Effect of Chalcogen Substitution on an Enzyme Mimetic for Deiodination of Thyroid Hormones

Iodothyronine deiodinases are selenoenzymes which regulate the thyroid hormone homeostasis by catalyzing the regioselective deiodination of thyroxine (T4). Synthetic deiodinase mimetics are important not only to understand the mechanism of enzyme catalysis, but also to develop therapeutic agents as abnormal thyroid hormone levels have implications in different diseases, such as hypoxia, myocardial infarction, critical illness, neuronal ischemia, tissue injury, and cancer. Described herein is that the replacement of sulfur/selenium atoms in a series of deiodinase mimetics by tellurium remarkably alters the reactivity as well as regioselectivity toward T4. The tellurium compounds reported in this paper represent the first examples of deiodinase mimetics which mediate sequential deiodination of T4 to produce all the hormone derivatives including T0 under physiologically relevant conditions.

Contrasting Electronic Requirements for C-H Binding and C-H Activation in d(6) Half-Sandwich Complexes of Rhenium and Tungsten

A computational study of the interaction half-sandwich metal fragments (metal=Re/W, electron count=d(6)), containing linear nitrosyl (NO+), carbon monoxide (CO), trifluorophosphine (PF3), N-heterocyclic carbene (NHC) ligands with alkanes are conducted using density functional theory employing the hybrid meta-GGA functional (M06). Electron deficiency on the metal increases with the ligand in the order NHC < CO < PF3 < NO+. Electron-withdrawing ligands like NO+ lead to more stable alkane complexes than NHC, a strong electron donor. Energy decomposition analysis shows that stabilization is due to orbital interaction involving charge transfer from the alkane to the metal. Reactivity and dynamics of the alkane fragment are facilitated by electron donors on the metal. These results match most of the experimental results known for CO and PF3 complexes. The study suggests activation of alkane in metal complexes to be facile with strong donor ligands like NHC. (C) 2015 Wiley Periodicals, Inc.

A carboxy terminal domain of the L protein of rinderpest virus possesses RNA triphosphatase activity - The first enzyme in the viral mRNA capping pathway

The large protein L of negative-sense RNA viruses is a multifunctional protein involved in transcription and replication of genomic RNA. It also possesses enzymatic activities involved in capping and methylation of viral mRNAs. The pathway for mRNA capping followed by the L protein of the viruses in the Morbillivirus genus has not been established, although it has been speculated that these viruses may follow the unconventional capping pathway as has been shown for some viruses of Rhabdoviridae family. We had earlier shown that the large protein L of Rinderpest virus expressed as recombinant L-P complex in insect cells as well as the ribonucleoprotein complex from purified virus possesses RNA triphosphatase (RTPase) and guanylyltransferase activities, in addition to RNA dependent RNA polymerase activity. In the present work, we demonstrate that RTPase as well as nucleoside triphosphatase (NTPase) activities are exhibited by a subdomain of the L protein in the C terminal region (a.a. 1640 1840). The RTPase activity depends absolutely on a divalent cation, either magnesium or manganese. Both the RTPase and NTPase activities of the protein show dual metal specificity. Two mutant proteins having alanine mutations in the glutamic acid residues in motif-A of the RTPase domain did not show RTPase activity, while exhibiting reduced NTPase activity suggesting overlapping active sites for the two enzymatic functions. The RTPase and NTPase activities of the L subdomain resemble those of the Vaccinia capping enzyme D1 and the baculovirus LEF4 proteins. (C) 2015 Elsevier Inc. All rights reserved.

Controlled Release of Salicylic Acid from Biodegradable Cross-Linked Polyesters

The purpose of this work was to develop a family of crosslinked poly(xylitol adipate salicylate)s with a wide range of tunable release properties for delivering pharmacologically active salicylic acid. The synthesis parameters and release conditions were varied to modulate polyester properties and to understand the mechanism of release. Varying release rates were obtained upon longer curing (35% in the noncured polymer to 10% in the cured polymer in 7 days). Differential salicylic acid loading led to the synthesis of polymers with variable cross-linking and the release could be tuned (100% release for the lowest loading to 30% in the highest loading). Controlled release was monitored by changing various factors, and the release profiles were dependent on the stoichiometric composition, pH, curing time, and presence of enzyme. The polymer released a combination of salicylic acid and disalicylic acid, and the released products were found to be nontoxic. Minimal hemolysis and platelet activation indicated good blood compatibility. These polymers qualify as ``bioactive'' and ``resorbable'' and can, therefore, find applications as immunomodulatory resorbable biomaterials with tunable release properties.