Characterization of a Negative cis-Acting DNA Element Regulating the Transcription of CYP2B1/B2 Gene in Rat Liver

The region -160 to -127 nt of the upstream of CYP-2B1/B2 gene has been found to function as a negative cis-acting element on the basis of DNase-I footprint and gel mobility shift assays as well as cell-free transcriptional assays using Bal-31 mutants. A reciprocal relationship in the interaction of the negative and the recently characterized positive elements with their respective protein factors has been found under repressed and induced conditions of the gene. The negative element also harbors the core glucocorticoid responsive sequence, TGTCCT. It is concluded that the negative element mediates the repressed state of the gene under the uninduced condition and also mediates the repressive effect of dexamethasone, when given along with the inducer phenobarbitone in rats. Dexamethasone is able to antagonize the effects of phenobarbitone at as low a concentration as 100 mu g/kg body wt in these animals. (C) 1995 Academic Press,Inc.

Interaction of sheep liver apo-serine hydroxymethyltransferase with pyridoxal-5'-phosphate: A physicochemical, kinetic, and thermodynamic study

Sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) is a homotetramer of M(r) 213,000 requiring pyridoxal-5'-phosphate (PLP) as cofactor, Removal of PLP from the holoenzyme converted the enzyme to the apo form which, in addition to being inactive, was devoid of the characteristic absorption spectrum. Upon the addition of PLP to the apoenzyme, complete activity was restored and the visible absorption spectrum with a maximum at 425 nm was regained. The interaction of PLP with the apoenzyme revealed two phases of reaction with pseudo-first-order rate constants of 20 +/- 5 s(-1) and 12.2 +/- 2.0 x 10(-3) s(-1), respectively. However, addition of PLP to the apoenzyme did not cause gross conformational changes as evidenced by circular dichroic and fluorescence spectroscopy. Although conformationally apoenzyme and holoenzyme were indistinguishable, they had distinct apparent melting temperatures of 51 +/- 2 and 58 +/- 2 degrees C, respectively, and the reconstituted holoenzyme was thermally as stable as the native holoenzyme. These results suggested that there was no apparent difference in the secondary structure of holoenzyme, apoenzyme, and reconstituted holoenzyme, However, sedimentation analysis of the apoenzyme revealed the presence of two peaks of S-20,S-w values of 8.7 +/- 0.5 and 5.7 +/- 0.3 S, respectively. A similar pattern was observed when the apoenzyme was chromatographed on a calibrated Sephadex G-150 column. The first peak corresponded to the tetrameric form (M(r) 200,000 +/- 15,000) while the second peak had a M(r) of 130,000 +/- 10,000. Reconstitution experiments revealed that only the tetrameric form of the apoenzyme could be converted into an active holoenzyme while the dimeric form could not be reconstituted into an active enzyme. These results demonstrate that PLP plays an important role in maintaining the structural integrity of the enzyme by preventing the dissociation of the enzyme into subunits, in addition to its function in catalysis. (C) 1996 Academic Press, Inc.

Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50kDa protein) to the membrane, present in the 58kDa-protein appears to be involved in mediating the inhibition by silatrane.

Experimental results of an activated carbon-HFC 134a adsorption cooling system for thermal management of electronics

Results of performance measurement of a small cooling capacity laboratory model of an adsorption refrigeration system for thermal management of electronics are compiled. This adsorption cooler was built with activated carbon as the adsorbent and HFC 134a as the refrigerant to produce a cooling capacity under 5 W using waste heat up to 90 degrees C. The thermal compression process is obtained from an ensemble of four solid sorption compressors. Parametric study was conducted with cycle times of 16 and 20 min, heat source temperatures from 73 to 87 degrees C and cooling loads from 3 to 4.9W. Overall system performance is analyzed using two indicators, namely, cooling effectiveness and normalized exergetic efficiency. (C) 2011 Elsevier Ltd. All rights reserved.

Ultrafast solvation dynamics in water: Isotope effects and comparison with experimental results

A detailed theoretical study of solvation dynamics in water is presented. The motivation of the present study comes from the recent experimental observation that the dynamics of solvation of an ion in water is ultrafast and the solvation time correlation function decays with a time constant of about 55 fs. The slower decay in the long time can be described by a sum of two exponentials with time constants equal to 126 and 880 fs. The molecular theory (developed earlier) predicts a time constant equal to 52 fs for the initial Gaussian decay and time constants equal to 134 and 886 fs for the two exponential components at the long time. This nearly perfect agreement is obtained by using the most detailed dynamical information available in the literature. The present study emphasizes the importance of the intermolecular vibrational band originating from the O...O stretching mode of the O�H...O units in the initial dynamics and raises several interesting questions regarding the nature of the decay of this mode. We have also studied the effects of isotope substitution on solvation dynamics. It is predicted that a significant isotope effect may be observed in the long time. The experimental results have also been compared with the prediction of the dynamic mean spherical approximation (DMSA); the agreement is not satisfactory at the long time. It is further found that the molecular theory and the DMSA lead to virtually identical results if the translational modes of the solvent molecules are neglected in the former. DMSA has also been used to investigate the dynamics of solvation of a dipolar solute in water. It is found that the dynamics of dipolar solvation exhibit features rather different from those of ion solvation. © 1995 American Institute of Physics.

Graphics Processing Units for the Real-time Linear Elastostatic Simulation of Liver

Biomedical engineering solutions like surgical simulators need High Performance Computing (HPC) to achieve real-time performance. Graphics Processing Units (GPUs) offer HPC capabilities at low cost and low power consumption. In this work, it is demonstrated that a liver which is discretized by about 2500 finite element nodes, can be graphically simulated in realtime, by making use of a GPU. Present work takes into consideration the time needed for the data transfer from CPU to GPU and back from GPU to CPU. Although behaviour of liver is very complicated, present computer simulation assumes linear elastostatics. One needs to use the commercial software ANSYS to obtain the global stiffness matrix of the liver. Results show that GPUs are useful for the real-time graphical simulation of liver, which in turn is needed in simulators that are used for training surgeons in laparoscopic surgery. Although the computer simulation should involve rendering also, neither rendering, nor the time needed for rendering and displaying the liver on a screen, is considered in the present work. The present work is just a demonstration of a concept; the concept is not really implemented and validated. Future work is to develop software which can accomplish real-time and very realistic graphical simulation of liver, with rendered image of liver on the screen changing in real-time according to the position of the surgical tool tip approximated as the mouse cursor in 3D.

Non-viral ex vivo hepatic gene transfer by in situ lipofection of liver and intraperitoneal transplantation of hepatocytes

Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal transplantation is a simple and cost-effective strategy of introducing genetically modified hepatocytes into liver. Thus, in situ lipofection of liver and intraperitoneal transfer of hepatocytes can be developed into a novel method of non-viral ex vivo gene transfer technique that has applications in the treatment of metabolic disorders of liver and hepatic gene therapy.

The role of lysine-256 in the structure and function of sheep liver recombinant serine hydroxymethyltransferase

The active site lysine residue, K256, involved in Schiffs base linkage with pyridoxal-5'-phosphate (PEP) in sheep liver recombinant serine hydroxymethyltransferase (rSHMT) was changed to glutamine or arginine by site-directed mutagenesis. The purified K256Q and K256R SHMTs had less than 0.1% of catalytic activity with serine and H(4)folate as substrates compared to rSHMT. The mutant enzymes also failed to exhibit the characteristic visible absorbance spectrum (lambda(max) 425 nm) and did not produce the quinonoid intermediate (lambda(max) 495 nm) upon the addition of glycine and H(4)folate. The mutant enzymes were unable to catalyze aldol cleavage of beta-phenylserine and transamination of D-alanine. These results suggested that the mutation of the lysine had resulted in the inability of the enzyme to bind to the cofactor. Therefore, the K256Q SHMT was isolated as a dimer and the K256R SHMT as a mixture of dimers and tetramers which were converted to dimers slowly. On the other hand, rSHMT was stable as a tetramer for several months, further confirming the role of PLP in maintenance of oligomeric structure. The mutant enzymes also failed to exhibit the increased thermal stability upon the addition of serine, normally observed with rSHMT. The enhanced thermal stability has been attributed to a change in conformation of the enzyme from open to closed form leading to reaction specificity. The mutant enzymes were unable to undergo this conformational change probably because of the absence of bound cofactor.

Halogen Bonding in 2,5-Dichloro-1,4-benzoquinone: Insights from Experimental and Theoretical Charge Density Analysis

Experimental charge density distribution in 2, 5-dichloro-1, 4-benzoquinone has been carried out using high resolution X-ray diffraction data at 90 K to quantitatively evaluate the nature of C-Cl center dot center dot center dot O=C halogen bond in molecular crystals. Additionally, the halogen bond is studied from geometrical point of view and the same has been visualized using Hirshfeld surface analysis. The obtained results from experimental charge density analysis are compared with periodic quantum calculations using B3LYP 6-31G(d,p) level of theory. The topological values at bond critical point, three-dimensional static deformation density features and electrostatic potential isosurfaces unequivocally establish the attractive nature of C-Cl center dot center dot center dot O=C halogen bond in crystalline lattice.

Role of indenter angle on the plastic deformation underneath a sharp indenter and on representative strains: An experimental and numerical study

The subsurface microhardness mapping technique of Chaudhri was utilized to determine the shape, size and distribution of plastic strain underneath conical indenters of varying semi-apex angles, alpha (55 degrees, 65 degrees and 75 degrees). Results show that the elastic-plastic boundary under the indenters is elliptical in nature, contradicting the expanding cavity model, and the ellipticity increases with alpha. The maximum plastic strain immediately under the indenter was found to decrease with increasing alpha. Complementary finite-element analysis was conducted to examine the ability of simulations to capture the experimental observations. A comparison of computational and experimental results indicates that the plastic strain distributions as well as the maximum strains immediately beneath the indenter do not match, suggesting that simulation of sharp indentation requires further detailed studies for complete comprehension. Representative strains, epsilon(r), evaluated as the volume-average strains within the elastic-plastic boundary, decrease with increasing alpha and are in agreement with those estimated by using the dimensional analysis. (C) 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Metabolic disposition of a monoterpene ketone, piperitenone, in rats: Evidence for the formation of a known toxin, p-cresol

It was shown earlier that the monoterpene ketone, piperitenone (I) is one of the major metabolites of R-(+)-pulegone, a potent hepatotoxin, In the present studies, the metabolic disposition of piperitenone (I) was examined in rats. Piperitenone (I) was administered orally (400 mg/kg of the b. wt./day) to rats for 5 days, The following urinary metabolites were isolated and identified by various spectral analyses: p-cresol (VI), 6,7-dehydromenthofuran (III), p-mentha-1,3,5,8-tetraen-3-ol (IX), p-mentha-1,3, 5-friene-3, 8-diol (X), 5-hydroxypiperitenone (VIII), 7-hydroxypiperitenone (XI), 10-hydroxypiperitenone (XII), and 4-hydroxypiperitenone (VII). Incubation of piperitenone (I) with phenobarbital-induced rat liver microsomes in the presence of NADPH resulted in the formation of five metabolites which have been tentatively identified as metabolites III, VII, VIII, XI, XII, on the basis of gas chromatography retention time and gas chromatography-mass spectrometry analysis. Based on these results, a probable mechanism for the formation of p-cresol from piperitenone (I) via the intermediacy of metabolite III has been proposed.

Experimental validation of analytical performance models for IEEE 802.11 networks

We consider the simplest IEEE 802.11 WLAN networks for which analytical models are available and seek to provide an experimental validation of these models. Our experiments include the following cases: (i) two nodes with saturated queues, sending fixed-length UDP packets to each other, and (ii) a TCP-controlled transfer between two nodes. Our experiments are based entirely on Aruba AP-70 access points operating under Linux. We report our observations on certain non-standard behavior of the devices. In cases where the devices adhere to the standards, we find that the results from the analytical models estimate the experimental data with a mean error of 3-5%.

Experimental study and optimization of Plasma Actuators for Flow control in subsonic regime

Experimental study and optimization of Plasma Ac- tuators for Flow control in subsonic regime PRADEEP MOISE, JOSEPH MATHEW, KARTIK VENKATRAMAN, JOY THOMAS, Indian Institute of Science, FLOW CONTROL TEAM | The induced jet produced by a dielectric barrier discharge (DBD) setup is capable of preventing °ow separation on airfoils at high angles of attack. The ef-fect of various parameters on the velocity of this induced jet was studied experimentally. The glow discharge was created at atmospheric con-ditions by using a high voltage RF power supply. Flow visualization,photographic studies of the plasma, and hot-wire measurements on the induced jet were performed. The parametric investigation of the charac- teristics of the plasma show that the width of the plasma in the uniform glow discharge regime was an indication of the velocity induced. It was observed that the spanwise and streamwise overlap of the two electrodes,dielectric thickness, voltage and frequency of the applied voltage are the major parameters that govern the velocity and the extent of plasma.e®ect of the optimized con¯guration on the performance characteristics of an airfoil was studied experimentally.

Simulation and experimental study on compositional evolution of Li-Co in LiCoO(2) thin films during sputter deposition

The compositional evolution in sputter deposited LiCoO(2) thin films is influenced by process parameters involved during deposition. The electrochemical performance of these films strongly depends on their microstructure, preferential orientation and stoichiometry. The transport process of sputtered Li and Co atoms from the LiCoO(2) target to the substrate, through Ar plasma in a planar magnetron configuration, was investigated based on the Monte Carlo technique. The effect of sputtering gas pressure and the substrate-target distance (d(st)) on Li/Co ratio, as well as, energy and angular distribution of sputtered atoms on the substrate were examined. Stable Li/Co ratios have been obtained at 5 Pa pressure and d(st) in the range 5 11 cm. The kinetic energy and incident angular distribution of Li and Co atoms reaching the substrate have been found to be dependent on sputtering pressure. Simulations were extended to predict compositional variations in films prepared at various process conditions. These results were compared with the composition of films determined experimentally using x-ray photoelectron spectroscopy (XPS). Li/Co ratio calculated using XPS was in moderate agreement with that of the simulated value. The measured film thickness followed the same trend as predicted by simulation. These studies are shown to be useful in understanding the complexities in multicomponent sputtering. (C) 2011 American Institute of Physics. doi:10.1063/1.3597829]

Phenobarbitone-mediated translocation of the cytosolic proteins interacting with the 5 '-proximal region of rat liver CYP2B1/B2 gene into the nucleus

The positive element (PE) (-69 to -98 bp) within the 5'-proximal region of the CYP2B1B2 gene (+1 to -179 bp) of rat liver is essential for phenobarbitone (PB) response and gives a single major complex with the rat liver cytosol in gel shift analysis. This complex corresponds to complex I (top) of the three complexes given by the nuclear extracts. PB treatment of rats leads to a decrease in complex I formation with the cytosol and PE and an increase in the same with the nuclear extract in gel shift analysis. Both the changes are counteracted by simultaneous okadaic acid administration. The nuclear protein giving rise to complex I has been isolated and has an M-r of 26 kDa. The cytosolic counterpart consists of two species, 26 and 28 kDa, as revealed by Southwestern blot analysis using labeled PE. It is concluded that PB treatment leads to the translocation accompanied by processing of the cytosolic protein species into the nucleus that requires protein dephosphorylation. It is suggested that PB may exert a global regulation on the transcription of many genes by modulating the phosphorylation status of different protein factors involved in transcriptional regulation. (C) 2002 Elsevier Science (USA).