Identification of Critical Residues of the Mycobacterial Dephosphocoenzyme A Kinase by Site-Directed Mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the c-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg2+ for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.

Interaction of Sesbania Mosaic Virus Movement Protein with VPg and P10: Implication to Specificity of Genome Recognition

Sesbania mosaic virus (SeMV) is a single strand positive-sense RNA plant virus that belongs to the genus Sobemovirus. The mechanism of cell-to-cell movement in sobemoviruses has not been well studied. With a view to identify the viral encoded ancillary proteins of SeMV that may assist in cell-to-cell movement of the virus, all the proteins encoded by SeMV genome were cloned into yeast Matchmaker system 3 and interaction studies were performed. Two proteins namely, viral protein genome linked (VPg) and a 10-kDa protein (P10) c v gft encoded by OFR 2a, were identified as possible interacting partners in addition to the viral coat protein (CP). Further characterization of these interactions revealed that the movement protein (MP) recognizes cognate RNA through interaction with VPg, which is covalently linked to the 59 end of the RNA. Analysis of the deletion mutants delineated the domains of MP involved in the interaction with VPg and P10. This study implicates for the first time that VPg might play an important role in specific recognition of viral genome by MP in SeMV and shed light on the possible role of P10 in the viral movement.

Thermomechanical generalization of pin joints in finite plates

The understanding of thermoelastic behaviour of joints is significant in order to ensure the integrity of large and complex structures exposed to a thermal environment, particularly in fields such as aerospace and nuclear engineering. Thermomechanical generalization of partial contact behaviour of a pin joint under combined in-plane mechanical loading and on-axis unidirectional heat flow has already been established by the authors for the analytically simpler domains of large plates. This paper successfully extends the on-going investigation to a single pin in a finite rectangular isotropic plate as a two-dimensional abstraction from a practical situation of a multipin fastener joint. The finite element method is used to analyse the joint problem under on-axis thermomechanical loading and unified load-contact relationships are established for a class of loading conditions.

A survey of indian logic from the point of view of computer science

Indian logic has a long history. It somewhat covers the domains of two of the six schools (darsanas) of Indian philosophy, namely, Nyaya and Vaisesika. The generally accepted definition of Indian logic over the ages is the science which ascertains valid knowledge either by means of six senses or by means of the five members of the syllogism. In other words, perception and inference constitute the subject matter of logic. The science of logic evolved in India through three ages: the ancient, the medieval and the modern, spanning almost thirty centuries. Advances in Computer Science, in particular, in Artificial Intelligence have got researchers in these areas interested in the basic problems of language, logic and cognition in the past three decades. In the 1980s, Artificial Intelligence has evolved into knowledge-based and intelligent system design, and the knowledge base and inference engine have become standard subsystems of an intelligent system. One of the important issues in the design of such systems is knowledge acquisition from humans who are experts in a branch of learning (such as medicine or law) and transferring that knowledge to a computing system. The second important issue in such systems is the validation of the knowledge base of the system i.e. ensuring that the knowledge is complete and consistent. It is in this context that comparative study of Indian logic with recent theories of logic, language and knowledge engineering will help the computer scientist understand the deeper implications of the terms and concepts he is currently using and attempting to develop.

Microstructural control in hot working of IN-718 superalloy using processing map

The hot-working characteristics of IN-718 are studied in the temperature range 900 °C to 1200 °C and strain rate range 0.001 to 100 s−1 using hot compression tests. Processing maps for hot working are developed on the basis of the strain-rate sensitivity variations with temperature and strain rate and interpreted using a dynamic materials model. The map exhibits two domains of dynamic recrystallization (DRX): one occurring at 950 °C and 0.001 s−1 with an efficiency of power dissipation of 37 pct and the other at 1200 °C and 0.1 s−1 with an efficiency of 40 pct. Dynamic recrystallization in the former domain is nucleated by the δ(Ni3Nb) precipitates and results in fine-grained microstructure. In the high-temperature DRX domain, carbides dissolve in the matrix and make interstitial carbon atoms available for increasing the rate of dislocation generation for DRX nucleation. It is recommended that IN-718 may be hot-forged initially at 1200 °C and 0.1 s−1 and finish-forged at 950 °C and 0.001 s−1 so that fine-grained structure may be achieved. The available forging practice validates these results from processing maps. At temperatures lower than 1000 °C and strain rates higher than 1 s−1 the material exhibits adiabatic shear bands. Also, at temperatures higher than 1150°C and strain rates more than 1s−1, IN-718 exhibits intercrystalline cracking. Both these regimes may be avoided in hotworking IN-718.

Microstructural control in hot working of IN-718 superalloy using processing map

The hot-working characteristics of IN-718 are studied in the temperature range 900 degrees C to 1200 degrees C and strain rate range 0.001 to 100 s(-1) using hot compression tests. Processing maps for hot working are developed on the basis of the strain-rate sensitivity variations with temperature and strain rate and interpreted using a dynamic materials model. The map exhibits two domains of dynamic recrystallization (DRX): one occurring at 950 degrees C and 0.001 s(-1) with an efficiency of power dissipation of 37 pct and the other at 1200 degrees C and 0.1 s(-1) with an efficiency of 40 pct. Dynamic recrystallization in the former domain is nucleated by the delta(Ni3Nb) precipitates and results in fine-grained microstructure. In the high-temperature DRX domain, carbides dissolve in the matrix and make interstitial carbon atoms available for increasing the rate of dislocation generation for DRX nucleation. It is recommended that IN-718 may be hot-forged initially at 1200 degrees C and 0.1 s(-1) and finish-forged at 950 degrees C and 0.001 s(-1) so that fine-grained structure may be achieved. The available forging practice validates these results from processing maps. At temperatures lower than 1000 degrees C and strain rates higher than 1 s(-1), the material exhibits adiabatic shear bands. Also, at temperatures higher than 1150 degrees C and strain rates more than 1 s(-1), IN-718 exhibits intercrystalline cracking. Both these regimes may be avoided in hot-working IN-718.

Processing of an α-2 aluminide alloy, Ti---24Al---11Nb

Compressive stress-strain curves have been generated over a range of temperatures (900-1100-degrees-C and strain rates (0.001-100 s-1) for two starting structures consisting of lath alpha2 and equiaxed alpha2 in a Ti-24Al-11Nb alloy. The data from these tests have been analysed in terms of a dynamic model for processing. The results define domains of strain rate and temperature in which dynamic recrystallization of alpha2 occurs for both starting structures. The rate controlling process for dynamic recrystallization is suggested to be cross-slip in the alpha2 phase. A region of processing instability has also been defined within which shear bands form in the lath structure. Recrystallization of the beta phase is shown to occur for different combinations of strain rate and temperature from those in which the alpha2 phase recrystallizes dynamically

Optimization of hot workability of an Al-Mg-Si alloy using processing maps

The hot workability of an Al-Mg-Si alloy has been studied by conducting constant strain-rate compression tests. The temperature range and strain-rate regime selected for the present study were 300-550 degrees C and 0.001-1 s(-1), respectively. On the basis of true stress data, the strain-rate sensitivity values were calculated and used for establishing processing maps following the dynamic materials model. These maps delineate characteristic domains of different dissipative mechanisms. Two domains of dynamic recrystallization (DRX) have been identified which are associated with the peak efficiency of power dissipation (34%) and complete reconstitution of as-cast microstructure. As a result, optimum hot ductility is achieved in the DRX domains. The strain rates at which DRX domains occur are determined by the second-phase particles such as Mg2Si precipitates and intermetallic compounds. The alloy also exhibits microstructural instability in the form of localized plastic deformation in the temperature range 300-350 degrees C and at strain rate 1 s(-1).

Mapping of B-cell epitopes of hemagglutinin protein of rinderpest virus

Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus FIN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination. (C) 2002 Elsevier Science (USA).

Expression pattern of Cubitus interruptus from the mulberry silkworm Bombyx mori in late developmental stages

A partial genomic clone of Bombyx mori homologue of the segment polarity gene Cubitus interruptus (BmCi), encoding the conserved zinc finger domain and harbouring two introns, has been characterized. BmCi was expressed in the silkglands of B. mori from embryonic to the late larval stages(3rd, 4th and 5th intermoults). The expression was confined to the anterior region of the middle silkglands, overlapping with the domain of sericin-2 expression and excluding the domains of Bm invected expression, namely the middle and posterior regions of the middle silkglands. In the wing discs, the expression was restricted to the anterior compartment, which increased from 4th to 5th larval intermoults and declined later in the pupal wing buds. In gonadal tissues (both ovaries and testes) BmCi was expressed from the larval to pupal stages. The transcripts were localized to the sperm tubes containing spermatogonia in the testis of Bombyx larvae. BmCi expression, however, was not detected in any of these tissues during the moulting stages. Expression of Ci in the wing discs and gonads is evolutionarily conserved, while the silkgland represents a novel domain. Our results imply that BmCi is involved in the specification and maintenance of micro-compartment identity within the middle silkglands.

String-like propagation of the 5-coordinated defect state in supercooled water: molecular origin of dynamic and thermodynamic anomalies

We find that at low temperature water, large amplitude (similar to 60 degrees) rotational jumps propagate like a string, with the length of propagation increasing with lowering temperature. The strings are formed by mobile 5-coordinated water molecules which move like a Glarum defect (J. Chem. Phys., 1960, 33, 1371), causing water molecules on the path to change from 4-coordinated to 5-coordinated and again back to 4-coordinated water, and in the process cause the tagged water molecule to jump, by following essentially the Laage-Hynes mechanism (Science, 2006, 311, 832-835). The effects on relaxation of the propagating defect causing large amplitude jumps are manifested most dramatically in the mean square displacement (MSD) and also in the rotational time correlation function of the O-H bond of the molecule that is visited by the defect (transient transition to the 5-coordinated state). The MSD and the decay of rotational time correlation function, both remain quenched in the absence of any visit by the defect, as postulated by Glarum long time ago. We establish a direct connection between these propagating events and the known thermodynamic and dynamic anomalies in supercooled water. These strings are found largely in the regions that surround the relatively rigid domains of 4-coordinated water molecules. The propagating strings give rise to a noticeable dynamical heterogeneity, quantified here by a sharp rise in the peak of the four-point density response function, chi(4)(t). This dynamics heterogeneity is also responsible for the breakdown of the Stokes-Einstein relation.

Design and Characterization of Stabilized Derivatives of Human CD4D12 and CD4D1

CD4 is present on the surface of T-lymphocytes and is the primary cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. A construct consisting of the first two domains of CD4 (CD4D12) is folded and binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192-16202]. We now report the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L511/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. Two mutations, G6A and V86L in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC(50)s as 4-domain CD4. These stabilized derivatives of human CD4 can be useful starting points for the design of other more complex viral entry inhibitors.

On Convergence of Differential Evolution Over a Class of Continuous Functions With Unique Global Optimum

Differential evolution (DE) is arguably one of the most powerful stochastic real-parameter optimization algorithms of current interest. Since its inception in the mid 1990s, DE has been finding many successful applications in real-world optimization problems from diverse domains of science and engineering. This paper takes a first significant step toward the convergence analysis of a canonical DE (DE/rand/1/bin) algorithm. It first deduces a time-recursive relationship for the probability density function (PDF) of the trial solutions, taking into consideration the DE-type mutation, crossover, and selection mechanisms. Then, by applying the concepts of Lyapunov stability theorems, it shows that as time approaches infinity, the PDF of the trial solutions concentrates narrowly around the global optimum of the objective function, assuming the shape of a Dirac delta distribution. Asymptotic convergence behavior of the population PDF is established by constructing a Lyapunov functional based on the PDF and showing that it monotonically decreases with time. The analysis is applicable to a class of continuous and real-valued objective functions that possesses a unique global optimum (but may have multiple local optima). Theoretical results have been substantiated with relevant computer simulations.

Design of conjugate, conjoined shapes and tilings using topology optimization

We present a method for obtaining conjugate, conjoined shapes and tilings in the context of the design of structures using topology optimization. Optimal material distribution is achieved in topology optimization by setting up a selection field in the design domain to determine the presence/absence of material there. We generalize this approach in this paper by presenting a paradigm in which the material left out by the selection field is also utilised. We obtain conjugate shapes when the region chosen and the region left-out are solutions for two problems, each with a different functionality. On the other hand, if the left-out region is connected to the selected region in some pre-determined fashion for achieving a single functionality, then we get conjoined shapes. The utilization of the left-out material, gives the notion of material economy in both cases. Thus, material wastage is avoided in the practical realization of these designs using many manufacturing techniques. This is in contrast to the wastage of left-out material during manufacture of traditional topology-optimized designs. We illustrate such shapes in the case of stiff structures and compliant mechanisms. When such designs are suitably made on domains of the unit cell of a tiling, this leads to the formation of new tilings which are functionally useful. Such shapes are not only useful for their functionality and economy of material and manufacturing, but also for their aesthetic value.

Smart polymer mediated purification and recovery of active proteins from inclusion bodies

Obtaining correctly folded proteins from inclusion bodies of recombinant proteins expressed in bacterial hosts requires solubilization with denaturants and a refolding step. Aggregation competes with the second step. Refolding of eight different proteins was carried out by precipitation with smart polymers. These proteins have different molecular weights, different number of disulfide bridges and some of these are known to be highly prone to aggregation. A high throughput refolding screen based upon fluorescence emission maximum around 340 nm (for correctly folded proteins) was developed to identify the suitable smart polymer. The proteins could be dissociated and recovered after the refolding step. The refolding could be scaled up and high refolding yields in the range of 8 mg L-1 (for CD4D12, the first two domains of human CD4) to 58 mg L-1 (for malETrx, thioredoxin fused with signal peptide of maltose binding protein) were obtained. Dynamic light scattering (DLS) showed that polymer if chosen correctly acted as a pseuclochaperonin and bound to the proteins. It also showed that the time for maximum binding was about 50 min which coincided with the time required for incubation (with the polymer) before precipitation for maximum recovery of folded proteins. The refolded proteins were characterized by fluorescence emission spectra, circular dichroism (CD) spectroscopy, melting temperature (T-m), and surface hydrophobicity measurement by ANS (8-anilinol-naphthalene sulfonic acid) fluorescence. Biological activity assay for thioredoxin and fluorescence based assay in case of maltose binding protein (MBP) were also carried out to confirm correct refolding. (C) 2012 Elsevier B.V. All rights reserved.