Mapping the common antigenic determinants in avidin and streptavidin

An epitope scan analysis of the whole sequence of avidin and core streptavidin using polyclonal antibodies to these two antigens reveal the presence of multiple common epitopes in both the proteins. These antigenic determinants consist mostly of either identical or similar residues. The antibody recognition sites in both antigens are shown to be localized to homologous regions.

Design and characterization of in-plane MEMS yaw rate sensor

In this paper, we present the design and characterization of a vibratory yaw rate MEMS sensor that uses in-plane motion for both actuation and sensing. The design criterion for the rate sensor is based on a high sensitivity and low bandwidth. The required sensitivity of the yawrate sensor is attained by using the inplane motion in which the dominant damping mechanism is the fluid loss due to slide film damping i.e. two-three orders of magnitude less than the squeeze-film damping in other rate sensors with out-of-plane motion. The low bandwidth is achieved by matching the drive and the sense mode frequencies. Based on these factors, the yaw rate sensor is designed and finally realized using surface micromachining. The inplane motion of the sensor is experimentally characterized to determine the sense and the drive mode frequencies, and corresponding damping ratios. It is found that the experimental results match well with the numerical and the analytical models with less than 5% error in frequencies measurements. The measured quality factor of the sensor is approximately 467, which is two orders of magnitude higher than that for a similar rate sensor with out-of-plane sense direction.

In situ synthesis and characterization of polyaniline-CaCu3Ti4O12 nanocrystal composites

High dielectric constant (ca. 2.4 x 10(6) at 1 kHz) nanocomposite of polyaniline (PANI)/CaCu3Ti4O12 (CCTO) was synthesized using a simple procedure involving in situ polymerization of aniline in dil. HCl. The PANI and the composite were subjected to X-ray diffraction, Fourier transform infrared, thermo gravimetric, scanning electron microscopy and transmission electron microscopy analyses. The presence of the nanocrystallites of CCTO embedded in the nanofibers of PANI matrix was established by TEM. Frequency dependent characteristics of the dielectric constant. dielectric loss and AC conductivity were studied for the PANI and the composites. The dielectric constant increased as the CCTO content increased in PANI but decreased with increasing frequency (100 Hz-1 MHz) of measurement. The dielectric loss was two times less than the value obtained for pure PANI around 100 Hz. The AC conductivity increased slightly up to 2 kHz as the CCTO content increased in the PANI which was attributed to the polarization of the charge carriers.

Sol-Gel Template Synthesis, Structural Characterization and Growth Mechanism of Barium Zirconate Nanotubes

In this paper, we report the synthesis of barium zirconate, BaZrO3, (BZ) nanotubes fabricated by the modified sol-gel method within the nanochannels of anodic aluminum oxide (AAO) templates. The morphology, structure, and composition of as prepared nanotubes were characterized by means of X-ray diffraction (XRD), field emission scanning electron microscope (FE-SEM), transmission electron microscope (TEM), selected-area electron diffraction ( SAED), high resolution TEM (HRTEM) and energy-dispersive X-ray spectroscopy (EDX). The results of XRD and SAED indicated that postannealed (at 650 degrees C for 1 h) BZ nanotubes (BZNTs) exhibited a polycrystalline cubic perovskite crystal structure. SEM and TEM analysis revealed that BZNTs possessed a uniform length and diameter (similar to 200 nm) and the thickness of the wall of the BZNTs was about 20 nm. Y-junctions, multiple branching and typical T-junctions were also observed in some BZNTs. EDX analysis demonstrated that stoichiometric BaZrO3 was formed. HRTEM image confirmed that the obtained BZNTs were composed of nanoparticles in the range of 5-10 nm. The possible formation mechanism of BZNTs was discussed.

Molecular Characterization of Sclerospora graminicola, the Incitant of Pearl Millet Downy Mildew Using ISSR Markers

The DNA polymorphism among 22 isolates of Sclerospora graminicola, the causal agent of downy mildew disease of pearl millet was assessed using 20 inter simple sequence repeats (ISSR) primers. The objective of the study was to examine the effectiveness of using ISSR markers for unravelling the extent and pattern of genetic diversity in 22 S. graminicola isolates collected from different host cultivars in different states of India. The 19 functional ISSR primers generated 410 polymorphic bands and revealed 89% polymorphism and were able to distinguish all the 22 isolates. Polymorphic bands used to construct an unweighted pair group method of averages (UPGMA) dendrogram based on Jaccard's co-efficient of similarity and principal coordinate analysis resulted in the formation of four major clusters of 22 isolates. The standardized Nei genetic distance among the 22 isolates ranged from 0.0050 to 0.0206. The UPGMA clustering using the standardized genetic distance matrix resulted in the identification of four clusters of the 22 isolates with bootstrap values ranging from 15 to 100. The 3D-scale data supported the UPGMA results, which resulted into four clusters amounting to 70% variation among each other. However, comparing the two methods show that sub clustering by dendrogram and multi dimensional scaling plot is slightly different. All the S. graminicola isolates had distinct ISSR genotypes and cluster analysis origin. The results of ISSR fingerprints revealed significant level of genetic diversity among the isolates and that ISSR markers could be a powerful tool for fingerprinting and diversity analysis in fungal pathogens.

Development and characterization of lysine based tripeptide analogues as inhibitors of Sir2 activity

Sirtuins are NAD(+) dependent deacetylases that modulate various essential cellular functions. Development of peptide based inhibitors of Sir2s would prove useful both as pharmaceutical agents and as effectors by which downstream cellular alterations can be monitored. Click chemistry that utilizes Huisgen's 1,3-dipolar cycloaddition permits attachment of novel modifications onto the side chain of lysine. Herein, we report the synthesis of peptide analogues prepared using click reactions on N epsilon-propargyloxycarbonyl protected lysine residues and their characterization as inhibitors of Plasmodium falciparum Sir2 activity. The peptide based inhibitors exhibited parabolic competitive inhibition with respect to acetylated-peptide substrate and parabolic non-competitive inhibition with NAD(+) supporting the formation of EI2 and E.NAD(+).I-2 complexes. Cross-competition inhibition analysis with the non-competitive inhibitor nicotinamide (NAM) ruled out the possibility of the NAM-binding site being the second inhibitor binding site, suggesting the presence of a unique alternate pocket commodating the inhibitor. One of these compounds was also found to be a potent inhibitor of the intraerythrocytic growth of P. falciparum with 50% inhibitory concentration in the micromolar range.

Functional characterization of the precursor and spliced forms of RecA protein of Mycobacterium tuberculosis

The recA locus of pathogenic mycobacteria differs from that of nonpathogenic species because it contains large intervening sequences nested in the RecA homology region that are excised by an unusual protein-splicing reaction. In vivo assays indicated that Mycobacterium tuberculosis recA partially complemented Escherichia coli recA mutants for recombination and mutagenesis. Further, splicing of the 85 kDa precursor to 38 kDa MtRecA protein was necessary for the display of its activity, in vivo. To gain insights into the molecular basis for partial and lack of complementation by MtRecA and 85 kDa proteins, respectively, we purified both of them to homogeneity. MtRecA protein, but not the 85 kDa form, bound stoichiometrically to single-stranded DNA in the presence of ATP. MtRecA protein was cross-linked to 8-azidoadenosine 5'-triphosphate with reduced efficiency, and kinetic analysis of ATPase activity suggested that it is due to decreased affinity for ATP. In contrast, the 85 kDa form was unable to bind ATP, in the presence or absence of ssDNA and, consequently, was entirely devoid of ATPase activity. Molecular modeling studies suggested that the decreased affinity of MtRecA protein for ATP and the reduced efficiency of its hydrolysis might be due to the widening of the cleft which alters the hydrogen bonds and the contact area between the enzyme and the substrate and changes in the disposition of the amino acid residues around the magnesium ion and the gamma-phosphate. The formation of joint molecules promoted by MtRecA protein was stimulated by SSB when the former was added first. The probability of an association between the lack and partial levels of biological activity of RecA protein(s) to that of illegitimate recombination in pathogenic mycobacteria is considered.

Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1

The genome of the human pathogen Entamoeba histolytica, a primitive protist, contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1-encoded endonuclease catalyzed reaction, determined under steady-state and single-turnover conditions, revealed a significant burst phase followed by a slower steady-state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the K-m was 2.6 x 10-8 m and the k(cat) was 1.6 x 10-2 sec-1. For linear E. histolytica DNA substrate the K-m and k(cat) values were 1.3 x 10-8 m and 2.2 x 10-4 sec-1 respectively. Single-turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg2+ was required for activity, 60% activity was seen with Mn2+ and none with other divalent metal ions. Substitution of PDX12-14D (a metal-binding motif) with PAX(12-14)D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1-encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome.

Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line

1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to +40 mV, with. a dominating fast component and a small slower component. 3. The steady-state inactivation, h(infinity), was related to potential by a Boltzmann distribution, underlying thr ee states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by al? initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O reversible arrow I-1 reversible arrow I-2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m(x) model. However, the best-fitted value of x varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha(m) and beta(m), were determined. Their functional dependence on the membrane potential was investigated.

A fluctuation-based characterization of athermal phase transitions:Application to shape memory alloys

We employ a fluctuation-based technique to investigate the athermal component associated with martensite phase transition, which is a prototype of temperature-driven structural transformation. Statistically, when the phase transition is purely athermal, we find that the temporal sequence of avalanches under constant drive is insensitive to the drive rate. We have used fluctuations in electrical resistivity or noise in nickel titanium shape memory alloys in three different forms: a thin film exhibiting well-defined transition temperatures,a highly disordered film, and a bulk wire of rectangular cross-section. Noise is studied in the realm of dynamic transition,viz.while the temperature is being ramped, which probes into the kinetics of the transformation at real time scales,and could probably stand out as a promising tool for material testing in various other systems, including nanoscale devices.

Two highly unsymmetrical tetradentate (N3O) Schiff base copper(II) complexes: Template synthesis, structural characterization, magnetic and computational studies

Two new copper(II) complexes, [Cu-2(L-1)(2)](ClO4)(2) (1) and [Cu(L-2)(ClO4)] (2), of the highly unsymmetrical tetradentate (N3O) Schiff base ligands HL1 and HL2 (where HL1 = N-(2-hydroxyacetophenone)-bis-3-aminopropylamine and HL2 = N-(salicyldehydine)-bis-3-aminopropylamine) have been synthesised using a template method. Their single crystal X-ray structures show that in complex 1 two independent copper(II) centers are doubly bridged through sphenoxo-O atoms (O1A and O1B) of the two ligands and each copper atom is five-coordinated with a distorted square pyramidal geometry. The asymmetric unit of complex 2 consists of two crystallographically independe N-(salicylidene) bis(aminopropyl)amine-copper(II) molecules, A and B, with similar square pyramidal geometries. Cryomagnetic susceptibility measurements (5-300 K) on complex 1 reveal a distinct antiferromagnetic interaction with J=-23.6 cm(-1), which is substantiated by a DFT calculation (J=-27.6 cm(-1)) using the B3LYP functional. Complex 1, immobilized over highly ordered hexagonal mesoporous silica, shows moderate catalytic activity for the epoxidation of cyclohexene and styrene in the presence of TBHP as an oxidant.

Thermo-mechanical characterization of surface-micromachined microheaters using in-line digital holography

This paper describes the application of lensless in-line digital holographic microscopy (DHM) to carry out thermo-mechanical characterization of microheaters fabricated through PolyMUMPs three-layer polysilicon surface micromachining process and subjected to a high thermal load. The mechanical deformation of the microheaters on the electrothermal excitation due to thermal stress is analyzed. The numerically reconstructed holographic images of the microheaters clearly indicate the regions under high stress. A double-exposure method has been used to obtain the quantitative measurements of the deformations, from the phase analysis of the hologram fringes. The measured deformations correlate well with the theoretical values predicted by a thermo-mechanical analytical model. The results show that lensless in-line DHM with Fourier analysis is an effective method for evaluating the thermo-mechanical characteristics of MEMS components.

Novel 3-dimensional sixfold interpenetrating diamondoid networks of copper(I) coordination polymers of polypyridyl ligands - Syntheses,characterization and crystal structures

Two new coordination polymers [Cu(L-1)(2)](n)(ClO4)(n)center dot 2nH(2)O (1), [Cu(L-2)(2)](n)(ClO4)(n)center dot 2nH(2)O (2) of polydentate imine/pyridyl ligands, L-1 and L-2 with Cu(I) ion have been synthesized and characterized by single crystal X-ray diffraction studies, elemental analyses, IR' UV-vis and NMR spectroscopy. They represent 3-dimensional, sixfold interpenetrating diamondoid network structures having large pores of dimension, 35 x 21 angstrom(2) in 1 and 38 x 19 angstrom(2) in 2, respectively.

Identification and Characterization of a Novel Deoxyhypusine Synthase in Leishmania donovani

Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (N-epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.