Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica

The thermodynamics of tie binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites, There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5, 5'-Br-2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein, The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium, Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites, These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCI in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.

Encapsulation and release of rifampicin using poly(vinyl pyrrolidone)-poly (methacrylic acid) polyelectrolyte capsules

Poly(vinyl pyrrolidone) and poly(methacrylic acid) multilayer capsules based on hydrogen bonding have been prepared by the layer-by-layer approach and used to encapsulate and release rifampicin, an antituberculosis drug. Removal of silica core using a buffer of ammonium fluoride and hydrofluoric acid at about pH 3 was found to produce better capsules than hydrofluoric acid alone. An eight-layered capsule had a wall thickness of 20 rim. Maximum encapsulation was found to be about 86 mu g at 40 degrees C with 1 +/- 0.2 x 10(6) capsules. Release studies showed a burst kind of release and maximum release was obtained above pH 7 where the capsules disintegrate rapidly thereby releasing the drug in a short period. Interactions studies with Mycobacterium smegmatis showed that the capsules were cytocompatible and the released drug functioned with the same efficacy as the free drug.

Synthesis and Evaluation of a Novel Class of G-Quadruplex-Stabilizing Small Molecules Based on the 1,3-Phenylene-Bis(piperazinyl benzimidazole) System

Achieving stabilization of telomeric DNA in G-quadruplex conformation by Various organic compounds has been an important goal for the medicinal chemists seeking to develop new anticancer agents. Several compounds are known to stabilize G-quadruplexes. However, relatively few are known to induce their formation and/or alter the topology, of the preformed quadruplex DNA. Herein, four compounds having the 1,3-phenylene-bis(piperazinyl benzimidazole) unit as a basic skeleton have been synthesized, and their interactions with the 24-mer telomeric DNA sequences from Tetrahymena thermophilia d(T(2)G(4))(4) have been investigated using high-resolution techniques Such as circular dichroism (CD) spectropolarimetry, CD melting, emission spectroscopy, and polyacrylamide gel electrophoresis. The data obtained, in the presence of one of three ions (Li+, Na+, or K+), indicate that all the new compounds have a high affinity for G-quadruplex DNA, and the strength of the binding with G-quadruplex depends on (1) phenyl ring substitution, (ii) the piperazinyl side chain, and (iii) the type of monovalent cation present in the buffer. Results further Suggest that these compounds are able to abet the conversion of the Intramolecular quadruplex into parallel stranded intermolecular G-quadruplex DNA. Notably, these compounds are also capable of inducing and stabilizing the parallel stranded quadruplex from randomly structured DNA in the absence of any stabilizing cation. The kinetics of the structural changes Induced by these compounds could be followed by recording the changes in the CD signal as a function of time. The implications of the findings mentioned above are discussed in this paper.

Optical tweezer for probing erythrocyte membrane deformability

We report that the average rotation speed of optically trapped crenated erythrocytes is direct signature of their membrane deformability. When placed in hypertonic buffer, discocytic erythrocytes are subjected to crenation. The deformation of cells brings in chirality and asymmetry in shape that makes them rotate under the scattering force of a linearly polarized optical trap. A change in the deformability of the erythrocytes, due to any internal or environmental factor, affects the rotation speed of the trapped crenated cells. Here we show how the increment in erythrocyte membrane rigidity with adsorption of Ca++ ions can be exhibited through this approach.

Photocytotoxic Oxovanadium(IV) Complexes Showing Light-Induced DNA and Protein Cleavage Activity

Oxovanadium(IV) complexes [VO(L)(B)]Cl-2 (1-3), where L is bis(2-benzimidazolylmethyl)amine and B is 1,10-phenanthroline(phen),dipyrido[3,2-d:2',3'-f]quinoxaline(dpq) or dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been prepared, characterized, and their photo-induced DNA and protein cleavage activity studied. The photocytotoxicity of complex 3 has been studied using adenocarcinoma A549 cells, The phen complex 1, structurally characterized by single-crystal X-ray crystallography, shows the presence of a vanadyl group in six-coordinate VON5 coordination geometry. The ligands L and phen display tridentate and bidentate N-donor chelating binding modes, respectively. The complexes exhibit a d-d band near 740 nm in 15% DMF-Tris-HCl buffer (pH 7.2). The phen and dpq complexes display an irreversible cathodic cyclic voltammetric response near -0.8 V in 20% DMF-Tris-HCl buffer having 0.1 M KCl as supporting electrolyte. The dppz complex 3 exhibits a quasi-reversible voltammogram near -0.6 V (vs SCE) that is assignable to the V(IV)-V(III)couple. The complexes bind to calf thymus DNA giving binding constant values in the range of 6.6 x 10(4)-2.9 x 10(5) M-1. The binding site size, thermal melting and viscosity binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor ``chemical nuclease'' activity in dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes are efficient photocleavers of plasmid DNA in UV-A light of 365 nm via a mechanistic pathway that involves formation of both singlet oxygen and hydroxyl radicals. The complexes show significant photocleavage of DNA in near-IR light (>750 nm) via hydroxyl radical pathway. Among the three complexes, the dppz complex 3 shows significant BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via hydroxyl radical pathway. The dppz complex 3 also exhibits photocytotoxicity in non-small cell lung carcinoma/human lung adenocarcinoma A549 cells giving IC50 value of 17 mu M in visible light(IC50 = 175 mu M in dark).

Delay Optimal Scheduling in a Two-Hop Vehicular Relay Network

We study a scheduling problem in a wireless network where vehicles are used as store-and-forward relays, a situation that might arise, for example, in practical rural communication networks. A fixed source node wants to transfer a file to a fixed destination node, located beyond its communication range. In the absence of any infrastructure connecting the two nodes, we consider the possibility of communication using vehicles passing by. Vehicles arrive at the source node at renewal instants and are known to travel towards the destination node with average speed v sampled from a given probability distribution. Th source node communicates data packets (or fragments) of the file to the destination node using these vehicles as relays. We assume that the vehicles communicate with the source node and the destination node only, and hence, every packet communication involves two hops. In this setup, we study the source node's sequential decision problem of transferring packets of the file to vehicles as they pass by, with the objective of minimizing delay in the network. We study both the finite file size case and the infinite file size case. In the finite file size case, we aim to minimize the expected file transfer delay, i.e. expected value of the maximum of the packet sojourn times. In the infinite file size case, we study the average packet delay minimization problem as well as the optimal tradeoff achievable between the average queueing delay at the source node buffer and the average transit delay in the relay vehicle.

Expression, purification and X-ray analysis of 1,3-propanediol dehydrogenase (Aq_1145) from Aquifex aeolicus VF5

1,3-Propanediol dehydrogenase is an enzyme that catalyzes the oxidation of 1,3-propanediol to 3-hydroxypropanal with the simultaneous reduction of NADP(+) to NADPH. SeMet-labelled 1,3-propanediol dehydrogenase protein from the hyperthermophilic bacterium Aquifex aeolicus VF5 was overexpressed in Escherichia coli and purified to homogeneity. Crystals of this protein were grown from an acidic buffer with ammonium sulfate as the precipitant. Single-wavelength data were collected at the selenium peak to a resolution of 2.4 angstrom. The crystal belonged to space group P3(2), with unit-cell parameters a = b = 142.19, c = 123.34 angstrom. The structure contained two dimers in the asymmetric unit and was solved by the MR-SAD approach.

An analytical model for performance evaluation of multimedia applications over EDCA in an IEEE 802.11e WLAN

We extend the modeling heuristic of (Harsha et al. 2006. In IEEE IWQoS 06, pp 178 - 187) to evaluate the performance of an IEEE 802.11e infrastructure network carrying packet telephone calls, streaming video sessions and TCP controlled file downloads, using Enhanced Distributed Channel Access (EDCA). We identify the time boundaries of activities on the channel (called channel slot boundaries) and derive a Markov Renewal Process of the contending nodes on these epochs. This is achieved by the use of attempt probabilities of the contending nodes as those obtained from the saturation fixed point analysis of (Ramaiyan et al. 2005. In Proceedings ACM Sigmetrics, `05. Journal version accepted for publication in IEEE TON). Regenerative analysis on this MRP yields the desired steady state performance measures. We then use the MRP model to develop an effective bandwidth approach for obtaining a bound on the size of the buffer required at the video queue of the AP, such that the streaming video packet loss probability is kept to less than 1%. The results obtained match well with simulations using the network simulator, ns-2. We find that, with the default IEEE 802.11e EDCA parameters for access categories AC 1, AC 2 and AC 3, the voice call capacity decreases if even one streaming video session and one TCP file download are initiated by some wireless station. Subsequently, reducing the voice calls increases the video downlink stream throughput by 0.38 Mbps and file download capacity by 0.14 Mbps, for every voice call (for the 11 Mbps PHY). We find that a buffer size of 75KB is sufficient to ensure that the video packet loss probability at the QAP is within 1%.

Oxovanadium(IV) complexes of phenanthroline bases: the dipyridophenazine complex as a near-IR photocytotoxic agent

Oxovanadium(IV) complexes [VOCl(B)(2)]Cl (1-3) of phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d: 2', 3'-f] quinoxaline (dpq in 2) and dipyrido[3,2-a: 2', 3'-c] phenazine (dppz in 3), have been prepared, characterized and their DNA and protein binding, photo-induced DNA and protein cleavage activity andm photocytotoxicity have been studied. Complex 2, structurally characterized by X-ray crystallography, shows the presence of a vanadyl group in VOClN4 coordination geometry. The dpq ligand displays a chelating mode of binding with a N-donor site trans to the oxo-group. The chloride ligand is cis to the oxo-group. The one-electron paramagnetic complexes show a d-d band near 715 nm in 15% DMF-Tris-HCl buffer. The complexes are redox active exhibiting a V(IV)/V(III) redox couple within -0.5 to -0.7 V vs. SCE in 20% DMF-Tris-HCl/0.1 M KCl. The complexes bind to calf thymus (CT) DNA in the order: 3 (dppz) > 2 (dpq) > 1 (phen). The binding data reveal the groove and/or partial intercalative DNA binding nature of the complexes. The complexes show chemical nuclease'' activity in the dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide via a hydroxyl radical pathway. The dpq and dppz complexes are efficient photocleavers of DNA in UV-A light of 365 nm forming reactive singlet oxygen (O-1(2)) and hydroxyl radical ((OH)-O-center dot) species. Complexes 2 and 3 also show DNA cleavage activity in red light (> 750 nm) by an exclusive (OH)-O-center dot pathway. The complexes display a binding propensity to bovine serum albumin (BSA) protein giving K-BSA values in the range of 7.1 x 10(4)-1.8 x 10(5) M-1. The dppz complex 3 shows BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via (OH)-O-center dot pathway. The dppz complex 3 exhibits significant PDT effect in human cervical cancer HeLa cells giving IC50 values of 1.0 mu M and 12.0 mu M in UV-A and visible light, respectively (IC50 = > 100 mu M in the dark).

A Novel Bandwidth Enhancement Technique for Cascode Amplifier

In this paper, a new technique is presented to increase the bandwidth for a single stage amplifier. Usually, -3 dB bandwidth of single stage amplifier is in few MHz. High output impedance and subsequent capacitive loading decrease the bandwidth of amplifier. The presented technique uses a load which itself acts as bandwidth enhancer. This high speed amplifier is designed on 180 nm CMOS technology, operates at 2.5 V power supply. This amplifier is succeeded by an output buffer to achieve a better linearity, high output swing and required output impedance for matching.

Anaerobic DNA cleavage activity in red light and photocytotoxicity of (pyridine-2-thiol)cobalt(III) complexes of phenanthroline bases

Cobalt(III) complexes [Co(pnt)(B)(2)](NO3)(2) (1-3) of pyridine-2-thiol (pnt) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2',3'-c] phenazine (dppz in 3), have been prepared, characterized and their photo-induced anaerobic DNA cleavage activity studied. The crystal structure of 1a as mixed ClO4- and PF6- salt of 1 shows a (CoN5S)-N-III coordination geometry in which the pnt and phen showed N,S- and N,N-donor binding modes, respectively. The complexes exhibit Co(III)/Co(II) redox couple near -0.3 V (vs. SCE) in 20% DMF-Tris-HCl buffer having 0.1 M TBAP. The complexes show binding propensity to calf thymus DNA giving K-b values within 2.2 x 10(4)-7.3 x 10(5) M-1. Thermal melting and viscosity data suggest DNA surface and/or groove binding of the complexes. The complexes show significant anaerobic DNA cleavage activity in red light under argon atmosphere possibly involving sulfide anion radical or thiyl radical species. The DNA cleavage reaction under aerobic medium in red light is found to involve both singlet oxygen and hydroxyl radical pathways. The dppz complex 3 shows non-specific BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via both hydroxyl and singlet oxygen pathways. The dppz complex 3 exhibits photocytotoxicity in HeLa cervical cancer cells giving IC50 values of 767 nM and 19.38 mu M in UV-A light of 365 nm and in the dark, respectively. A significant reduction of the dark toxicity of the dppz base (IC50 = 8.34 mu M in dark) is observed on binding to the cobalt(III) center.

A Petri net model for evaluating packet buffering strategies in a network processor

Previous studies have shown that buffering packets in DRAM is a performance bottleneck. In order to understand the impediments in accessing the DRAM, we developed a detailed Petri net model of IP forwarding application on IXP2400 that models the different levels of the memory hierarchy. The cell based interface used to receive and transmit packets in a network processor leads to some small size DRAM accesses. Such narrow accesses to the DRAM expose the bank access latency, reducing the bandwidth that can be realized. With real traces up to 30% of the accesses are smaller than the cell size, resulting in 7.7% reduction in DRAM bandwidth. To overcome this problem, we propose buffering these small chunks of data in the on chip scratchpad memory. This scheme also exploits greater degree of parallelism between different levels of the memory hierarchy. Using real traces from the internet, we show that the transmit rate can be improved by an average of 21% over the base scheme without the use of additional hardware. Further, the impact of different traffic patterns on the network processor resources is studied. Under real traffic conditions, we show that the data bus which connects the off-chip packet buffer to the micro-engines, is the obstacle in achieving higher throughput.

Design, Synthesis, and DNA Binding Properties of Photoisomerizable Azobenzene−Distamycin Conjugates: An Experimental and Computational Study

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene−distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electron-withdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT)10CG)]2 DNA were performed to rationalize the nature of binding of these conjugates.

Iron(III) Schiff base complexes of arginine and lysine as netropsin mimics showing AT-selective DNA binding and photonuclease activity

Iron(III) complexes [Fe(L)(2)]Cl (1-3), where L is monoanionic N-salicylidene-arginine (sal-argH for 1), hydroxynaphthylidene-arginine (nap-argH for 2) and N-salicylidene-lysine (sal-lysH for 3), were prepared and their DNA binding and photo-induced DNA cleavage activity studied. Complex 3 as its hexafluorophosphate salt [Fe(sal-lysH)(2)](PF6)center dot 6H(2)O (3a) was structurally characterized by single crystal Xray crystallography. The crystals belonged to the triclinic space group P-1. The complex has two tridentate ligands in FeN2O4 coordination geometry with two pendant cationic amine moieties. Complexes 1 and 2 with two pendant cationic guanidinium moieties are the structural models for the antitumor antibiotics netropsin. The complexes are stable and soluble in water. They showed quasi-reversible Fe(III)/Fe(II) redox couple near 0.6 V in H2O-0.1 M KCl. The high-spin 3d(5)-iron(III) complexes with mu(eff) value of similar to 5.9 mu(B) displayed ligand-to-metal charge transfer electronic band near 500 mm in Tris-HCl buffer. The complexes show binding to Calf Thymus (CT) DNA. Complex 2 showed better binding propensity to the synthetic oligomer poly(dA)center dot poly(dT) than to CT-DNA or poly(dG)center dot poly(dC). All the complexes displayed chemical nuclease activity in the presence of 3-mercaptopropionic acid as a reducing agent and cleaved supercoiled pUC19 DNA to its nicked circular form. They exhibited photo-induced DNA cleavage activity in UV-A light and visible light via a mechanistic pathway that involves the formation of reactive hydroxyl radical species. (C) 2010 Elsevier Inc. All rights reserved.

Immobilization of Aspergillus oryzae alpha-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study, alpha-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions of immobilization. The immobilized alpha-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum temperature also shifted from 50 degrees C to 57 degrees C compared with soluble enzyme. Immobilized alpha-galactosidase was used in batch, repeated batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the fourth cycle. The performance of immobilized alpha-galactosidase was tested in a fluidized bed reactor at different flow rates and 86% reduction of RO in soymilk was obtained at 25 ml h(-1) flow rate. The study revealed that immobilized alpha-galactosidase in continuous mode is efficient in reduction of RO present in soymilk.