Experimental Investigation on Self-Compacting Self-Curing Concrete Incorporated with the Light Weight Aggregates

ABSTRACT Self-Compacting Concrete (SCC) flows around obstructions by its self-weight to fill entirely and self-consolidate (without any need for vibration), without any part of disconnection and chunking. The eradication of the need for consolidation leads to better quality concrete and substantial improvement of running conditions. The fresh state of SCC mixes will usually have huge amount of fine fillers, including cement, and produce excessively high compressive strength concrete. In order to overcome the workability problem Super Plasticizer (SP) and Viscosity Modifying Agent (VMA) are used. Here, the workability admixtures are fixed at a constant rate of 2% based on the weight of cement. This technique examines special applications in cases of bottleneck reinforced sections, rafts, tunnel linings, highly reinforced columns, underwater repairs, bridge piers and placements. LECA and Vermiculite have high porosity, and are added to concrete mixtures to create a lightweight concrete mix. An attempt has been made to develop a combination of self-compacting and self-curing concrete with each 5% and 10% of LECA and Vermiculite as a partial replacement to fine aggregate. As there was no proper mix design for the development of this modern concrete, the design has been carried out based on EFNARC specifications for the design strength of M40 grade concrete. The porosity of light weight aggregate provide source of water for internal curing of concrete which enhances concrete strength and durability. Based on more trials, it was noticed that the concrete with 10% of LECA and Vermiculite in individual provides good results.

Mechanical Behavior of Concrete Modified by Replacement of Cement by Rice Husk Ash

ABSTRACT Construction industry is in need of lump sum quantities of materials which has increased both their demand and price. The use of large quantities of cement leads to increasing CO2 emission and as a consequence, the greenhouse effect. Consumption of wastes and byproducts from various sources in the manufacture of concrete has gained a great deal of importance in present days. Various researches are currently being conducted concerning the use of such products in concrete. RHA is a carbon neutral green product. Lots of ways are being thought of for disposing them by making commercial use of this. Rice husk ash is a good super-pozzolan which can be used to make special concrete mixes. The rice husk ash has been taken for this present study due to its easy availability and effective pozzolonic properties that are expected to improve the mechanical strength properties of concrete. Concrete specimens were made for evaluation of Compressive, Split Tensile, Flexural strength and Stress-Strain Behavior of concrete. The tests were conducted at the age of 7 and 28 days. Generally all mixes containing RHA achieved better properties than the conventional mix without RHA. By the experimental investigation the recommendation is given for using optimum percentage of RHA in concrete.

Structural Behavior of SFRC Beams strengthened with GFRP Laminates: An Experimental and Analytical Investigation

ABSTRACT This paper presents the experimental results of a reinforced concrete beams (RC) strengthened with internal steel fibers (SF) and external glass fiber reinforced polymer laminates (GFRP). The research work studied the load carrying capacity, deformation, crack width and ductility of the reinforced concrete beams strengthened with different steel fiber ratios and steel fiber reinforced concrete beams strengthened with three different glass fiber reinforced polymer laminates of two different thickness. The experimental results clearly shows that incorporating steel fibers in to the reinforced concrete beams reduced the crack width and distribute the crack evenly and also increases the bonding between tension face of the beam with glass fiber reinforced polymer laminates. The results also shows that glass fiber reinforced polymer laminates strengthened steel fiber reinforced concrete beams increases the flexural strength and ductility as compared with unstrengthened counterpart. In addition to this experimental work, theoretical calculations were done to find the ultimate load carrying capacity of the beam tested, and also compared with the experimental results.

High-throughput screening assay for helicase enzymes.

We have developed a novel 96-well microtiter plate high-throughput screening filtration assay for the detection of helicase activity. In this paper we present data for detection of helicase activity of the UL5/8/52 helicase-primase complex from herpes simplex virus 1 (HSV1). The assay involves the detection of radioactively labeled oligonucleotide annealed to a single-stranded circular DNA following capture of the annealed complex on silica beads. We have screened over 200,000 samples containing small organic molecules and natural products and identified T157602, a two-amino thiazole, as a specific inhibitor of HSV replication.

Inhibition of herpes simplex virus replication by a 2-amino thiazole via interactions with the helicase component of the UL5-UL8-UL52 complex.

With the use of a high-throughput biochemical DNA helicase assay as a screen, T157602, a 2-amino thiazole compound, was identified as a specific inhibitor of herpes simplex virus (HSV) DNA replication. T157602 inhibited reversibly the helicase activity of the HSV UL5-UL8-UL52 (UL5/8/52) helicase-primase complex with an IC50 (concentration of compound that yields 50% inhibition) of 5 microM. T157602 inhibited specifically the UL5/8/52 helicase and not several other helicases. The primase activity of the UL5/8/52 complex was also inhibited by T157602 (IC50 = 20 microM). T157602 inhibited HSV growth in a one-step viral growth assay (IC90 = 3 microM), and plaque formation was completely prevented at concentrations of 25 to 50 microM T157602. Vero, human foreskin fibroblast (HFF), and Jurkat cells could be propagated in the presence of T157602 at concentrations exceeding 100 microM with no obvious cytotoxic effects, indicating that the window between antiviral activity and cellular toxicity is at least 33-fold. Seven independently derived T157602-resistant mutant viruses (four HSV type 2 and three HSV type 1) carried single base pair mutations in the UL5 that resulted in single amino acid changes in the UL5 protein. Marker rescue experiments demonstrated that the UL5 gene from T157602-resistant viruses conferred resistance to T157602-sensitive wild-type viruses. Recombinant UL5/8/52 helicase-primase complex purified from baculoviruses expressing mutant UL5 protein showed complete resistance to T157602 in the in vitro helicase assay. T157602 and its analogs represent a novel class of specific and reversible anti-HSV agents eliciting their inhibitory effects on HSV replication by interacting with the UL5 helicase.

1H-NMR detectable fatty acyl chain unsaturation in excised leiomyosarcoma correlate with grade and mitotic activity.

We report on the use of 1H-NMR two-dimensional total correlated spectroscopy (2D TOCSY) at 600 MHz for an ex vivo analysis of fatty acyl chain lipid in normal smooth muscle and a series of primary retroperitoneal leiomyosarcomas. These TOCSY spectra were used to identify and quantitate the methylene protons situated between unsaturated site protons (D) to those bordered by only one unsaturated site proton (C). The D/C cross-peak volume ratios determined for oleic (18:1), linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids were 0.0, 1.3, 2.7, and 4.0, respectively, suggesting that this ratio can be a measure of the degree of unsaturation for fatty acyl chains of lipids. The D/C cross-peak volume ratio was found to be proportional to the mean mitotic activity (r = 0.94) in nine smooth muscle tissues. These results suggest, that for leiomyosarcoma, the degree of fatty acyl unsaturation may be an important determinant of the metastatic potential of these tumors. Furthermore, application of TOCSY for the ex vivo study of smooth muscle tumors would potentially serve as a pathologist-independent and quantitative method for assessment of leiomyosarcoma grade and mitotic activity thereby rendering a more accurate staging of patients.

Ex vivo two-dimensional proton nuclear magnetic resonance spectroscopy of smooth muscle tumors: advantages of total correlated spectroscopy over homonuclear J-correlated spectroscopy.

Two-dimensional total correlated proton nuclear magnetic resonance spectroscopy (TOCSY) at 600 MHz was used for an ex vivo biochemical analysis of a benign human smooth muscle tumor (leiomyoma) and high grade malignant smooth muscle tumor (leiomyosarcoma). While there are a number of applications of proton nuclear magnetic resonance magnitude-mode two-dimensional correlated spectroscopy (COSY) to the ex vivo study of tissues and cells in the literature, to our knowledge this is the first application of TOCSY for the ex vivo study of biological tissue. Comparison of TOCSY and purged COSY data demonstrate the potential sensitivity advantages of the TOCSY method for the study of heterogeneous biological tissues. These TOCSY spectra were used to identify and quantitate a wide range of metabolites such as amino acids, peptides, triglycerides, phospholipid precursors and degradation products, bound fucose, and other saccharides. The leiomyosarcoma was found to have 5-fold higher levels of triglycerides and a 7-fold increase in the glycerophosphocholine:choline ratio compared to the leiomyoma. These metabolite changes may enhance membrane fluidity in the leiomyosarcoma compared to leiomyoma and thus may be of fundamental importance to cell motility, recognition, sarcoma tumorigenesis, and metastatic potential.

Solution structure of a POU-specific homeodomain: 3D-NMR studies of human B-cell transcription factor Oct-2.

The POU DNA-binding motif defines a conserved family of eukaryotic transcription factors involved in regulation of gene expression. This bipartite motif consists of an N-terminal POU-specific domain (POUs), a flexible linker, and a C-terminal POU-specific homeodomain (POUHD). Here we describe the solution structure of a POU-specific homeodomain. An NMR model is obtained from Oct-2, a human B-cell specific transcription factor which participates in the regulation of immunoglobulin genes. A fragment of Oct-2 containing POUHD and an adjoining linker was expressed in Escherichia coli and characterized by three-dimensional nuclear magnetic resonance (3D-NMR) spectroscopy. Complete 1H and 15N resonance assignment of the POUHD moiety is presented. The POUHD solution structure, as calculated by distance geometry and simulated annealing (DG/SA), is similar to that of canonical homeodomains. A salient difference between solution and crystal structures is observed in the C-terminal segment of alpha-helix 3 (the HTH recognition helix), which is not well ordered in solution. Because this segment presumably folds upon specific DNA binding, its flexibility in solution may reduce the intrinsic DNA affinity of POUHD in the absence of POUs.

The dimanganese(III,IV) oxidation state of catalase from Thermus thermophilus: electron nuclear double resonance analysis of water and protein ligands in the active site.

The 1H hyperfine tensors of the dimanganese(III,IV) oxidation state of the non-heme-type catalase enzyme from the thermophilic bacterium Thermus thermophilus have been measured by electron nuclear double resonance (ENDOR) spectroscopy at pH 6.5-9. These were compared to model dimanganese(III,IV) complexes possessing six-coordinate N4O2, N3O3, and O6 atom donor sets to each Mn and mu-oxo and mu-carboxylato bridging ligands. The lack of 14N hyperfine couplings in the enzyme suggests either O6 or O5N ligand donors to each Mn. Moreover, the two sigma coordination sites on Mn(III) directed at the dz2 orbital cannot be occupied by N ligands. The 1H ENDOR spectrum revealed two types of anisotropic tensors, attributable to two D2O-exchangeable protons on the basis of the magnitude of the electron paramagnetic resonance (EPR) line narrowing in D2O. All six of the 1H hyperfine couplings are proposed to arise from a single displaceable water molecule in the active site, on the basis of their reversible disappearance, upon incubation in D2O or by precipitation from ammonium sulfate, and by simulation of the 1H ENDOR spectrum. The Mn ions are coordinated predominantly by nonmagnetic O atoms lacking covalently bound protons in both alpha and beta positions. This implicates predominantly carboxylato-type ligands (Asp and Glu) and possibly a di-mu-oxo bridge between Mn ions. The latter is supported also by the presence of strong antiferromagnetic coupling. Comparison to other dimetalloproteins also possessing the four-helix bundle structural motif shows that the polyoxo(carboxylato) coordination in catalase differs significantly from the polyhistidine coordination adopted by the diiron(II,II) site in the O2-binding protein myohemerythrin, but resembles the polycarboxylato ligation adopted by the diiron(III,III) site of ribonucleotide reductase. The catalase 1H ENDOR spectrum is essentially identical to that for the exchangeable protons in the active site of the diiron(II,III) state of uteroferrin, an acid phosphatase [Doi et al. (1988) J. Biol. Chem. 263, 5757-5763], and also for a polycarboxylato complex possessing the Mn2(mu-O)2 core with H-bonded water ligands. The 1H ENDOR line shape in catalase could be simulated using a theoretical model suitable for multispin clusters. It treats the two Mn spins as point dipoles which are exchange-coupled. It includes both dipolar and isotropic ligand hyperfine couplings. Using this model, the position of the proton with the largest interaction could be located with respect to the Mn-Mn vector because of the extreme sensitivity of line shape to position.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.

Calcium limits substrate accessibility or reactivity at the manganese cluster in photosynthetic water oxidation.

The structural changes which the tetramanganese cluster responsible for catalyzing photosynthetic water oxidation undergoes upon calcium depletion of photosystem II membranes via the citrate extraction method has been further characterized. The modified multiline EPR signal which forms has been further identified with an S2' oxidation state. The increased number of hyperfine lines (at least 26) and 25% narrower hyperfine splittings from 55Mn versus the normal S2-state signal indicate a redistribution of spin density, most likely within a spin-coupled tetranuclear Mn cluster. A simpler binuclear Mn description for this signal can be eliminated. Slow conformational changes occur over 30 min which cause subtle changes in the hyperfine structure. Comparison to the modified multiline signals produced by Sr2+ replacement of Ca2+ and NH3-treated PSII reveal similarities suggestive of formation of the same spin S = 1/2 state. Substrate accessibility in the dark S1' state, measured as Mn2+ release upon incubation with NH2OH, is increased by 10-fold over calcium-containing PSII centers. Diphenylcarbazide, an efficient electron donor to Tyr-Z+ only in PSII membranes in which Mn is removed or dislocated, was found to donate electrons in Ca(2+)-depleted PSII, indicating altered accessibility or reactivity. These results suggest a possible "gatekeeper" role for Ca2+ in limiting access of substrate water to the Mn cluster. These changes are not due to release of the three extrinsic polypeptides of PSII which remain bound. Citrate treatment also causes partial air oxidation of the reaction-center Fe2+ ion, associated with the quinone electron acceptors. The resulting Fe3+ possesses an EPR signal at g = 4.37 arising from conversion to a rhombic symmetry ligand field.

Ca2+ depletion from the photosynthetic water-oxidizing complex reveals photooxidation of a protein residue.

A new intermediate in the water-oxidizing reaction has been observed in spinach photosystem II (PSII) membranes that are depleted of Ca2+ from the site which is conformationally coupled to the manganese cluster comprising the water-oxidizing complex (WOC). It gives rise to a recently identified EPR signal (symmetric line shape with width 163 +/- 5 G, g = 2.004 +/- 0.005), which forms in samples inhibited either by depletion of Ca2+ [Boussac, A., Zimmerman, J.-L., & 28, 8984-8989; Sivaraja, M., Tso, J., & Dismukes, G.C. (1989) Biochemistry 28 9459-9464] or by substitution of Cl- by F- (Baumgarten, Philo, and Dismukes, submitted for publication). Further characterization of this EPR signal has revealed the following: (1) it forms independently of the local structure of the PSII acceptors; (2) it arises from photooxidation of a PSII species that donates an electron to Tyr-Z+ or to the Mn cluster in competition with an exogenous donor (DPC); (3) the Curie temperature dependence of the intensity suggests an isolated doublet ground state, attributable to a spin S = 1/2 radical; (4) the electron spin orientation relaxes 1000-fold more rapidly than typical for a free radical, exhibiting a strong temperature dependence of P1/2 (half-saturation power approximately T3.4) and a broad inhomogeneous line width; (5) it yields an undetectable change in the magnetic susceptibility upon formation by a laser flash; (6) it disappears in parallel with release of Mn during reduction with NH2OH, indicating that it forms only in the presence of the modified Mn cluster. (ABSTRACT TRUNCATED AT 250 WORDS)

A calcium-specific site influences the structure and activity of the manganese cluster responsible for photosynthetic water oxidation.

EPR studies have revealed that removal of calcium using citric acid from the site in spinach photosystem II which is coupled to the photosynthetic O2-evolving process produces a structural change in the manganese cluster responsible for water oxidation. If done in the dark, this yields a modified S1' oxidation state which can be photooxidized above 250 K to form a structurally altered S2' state, as seen by formation of a "modified" multiline EPR signal. Compared to the "normal" S2 state, this new S2'-state EPR signal has more lines (at least 25) and 25% narrower 55Mn hyperfine splittings, indicative of disruption of the ligands to manganese. The calcium-depleted S2' oxidation state is greatly stabilized compared to the native S2 oxidation state, as seen by a large increase in the lifetime of the S2' EPR signal. Calcium reconstitution results in the reduction of the oxidized tyrosine residue 161YD+ (Em approximately 0.7-0.8 V, NHE) within the reaction center D1 protein in both the S1' and S2' states, as monitored by its EPR signal intensity. We attribute this to reduction by Mn. Thus a possible structural role which calcium plays is to bring YD+ into redox equilibrium with the Mn cluster. Photooxidation of S2' above 250 K produces a higher S state (S3 or S4) having a new EPR signal at g = 2.004 +/- 0.003 and a symmetric line width of 163 +/- 3 G, suggestive of oxidation of an organic donor, possibly an amino acid, in magnetic contact with the Mn cluster. This EPR signal forms in a stoichiometry of 1-2 relative to YD+.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification by ENDOR of Trp191 as the free-radical site in cytochrome c peroxidase compound ES.

The chemical identity of the amino acid free-radical site that represents one of the two oxidizing equivalents stored in the H2O2-oxidized intermediate (compound ES) of the mitochondrial heme enzyme, cytochrome c peroxidase (CcP) has been sought for almost a quarter of a century. Site-directed mutagenesis alone cannot yield this answer. Low-temperature 35-gigahertz (Q-band) electron nuclear double resonance (ENDOR) spectroscopy was used to examine compound ES prepared from proteins containing specifically deuterated methionine or tryptophan, as well as the amino acid replacement Trp51----Phe. The results definitely identify the site of the radical in compound ES as tryptophan, most likely Trp191.

Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters.

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Mössbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.