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.

Particle filters for structural system identification using multiple test and sensor data: A combined computational and experimental study

The problem of structural system identification when measurements originate from multiple tests and multiple sensors is considered. An offline solution to this problem using bootstrap particle filtering is proposed. The central idea of the proposed method is the introduction of a dummy independent variable that allows for simultaneous assimilation of multiple measurements in a sequential manner. The method can treat linear/nonlinear structural models and allows for measurements on strains and displacements under static/dynamic loads. Illustrative examples consider measurement data from numerical models and also from laboratory experiments. The results from the proposed method are compared with those from a Kalman filter-based approach and the superior performance of the proposed method is demonstrated. Copyright (C) 2009 John Wiley & Sons, Ltd.

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion Of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.

Identification of Local Conformational Similarity in Structurally Variable Regions of Homologous Proteins Using Protein Blocks

Structure comparison tools can be used to align related protein structures to identify structurally conserved and variable regions and to infer functional and evolutionary relationships. While the conserved regions often superimpose well, the variable regions appear non superimposable. Differences in homologous protein structures are thought to be due to evolutionary plasticity to accommodate diverged sequences during evolution. One of the kinds of differences between 3-D structures of homologous proteins is rigid body displacement. A glaring example is not well superimposed equivalent regions of homologous proteins corresponding to a-helical conformation with different spatial orientations. In a rigid body superimposition, these regions would appear variable although they may contain local similarity. Also, due to high spatial deviation in the variable region, one-to-one correspondence at the residue level cannot be determined accurately. Another kind of difference is conformational variability and the most common example is topologically equivalent loops of two homologues but with different conformations. In the current study, we present a refined view of the ``structurally variable'' regions which may contain local similarity obscured in global alignment of homologous protein structures. As structural alphabet is able to describe local structures of proteins precisely through Protein Blocks approach, conformational similarity has been identified in a substantial number of `variable' regions in a large data set of protein structural alignments; optimal residue-residue equivalences could be achieved on the basis of Protein Blocks which led to improved local alignments. Also, through an example, we have demonstrated how the additional information on local backbone structures through protein blocks can aid in comparative modeling of a loop region. In addition, understanding on sequence-structure relationships can be enhanced through our approach. This has been illustrated through examples where the equivalent regions in homologous protein structures share sequence similarity to varied extent but do not preserve local structure.

A novel method for mapping assembled epitopes in batches: Identification of three epitopes at the receptor binding region of human chorionic gonadotropin

Identification of conformation-specific epitopes of hCG beta has been done using a simple batch method, Chemically or enzymatically-modified hCG beta has been prepared in a batch and the effect of modifications on the integrity of different epitope regions has been investigated in a quantitative manner using monoclonal antibodies (MAbs) immobilized on plastic tubes from culture supernatants. Based on the extent of damage done to different regions by different modifications, three conformation-specific epitopes of hCG beta have been identified. The method has been shown to have important advantages over the existing methods on many considerations, Using this approach, these epitopes have been shown to be at/near the receptor-binding region.

FIR system identification based on subspaces of a higher order cumulant matrix

A new linear algebraic approach for identification of a nonminimum phase FIR system of known order using only higher order (>2) cumulants of the output process is proposed. It is first shown that a matrix formed from a set of cumulants of arbitrary order can be expressed as a product of structured matrices. The subspaces of this matrix are then used to obtain the parameters of the FIR system using a set of linear equations. Theoretical analysis and numerical simulation studies are presented to characterize the performance of the proposed methods.

Automatic identification of modal damping from Floquet analysis

A practical method is proposed to identify the mode associated with the frequency part of the eigenvalue of the Floquet transition matrix (FTM). From the FTM eigenvector, which contains the states and their derivatives, the ratio of the derivative and the state corresponding to the largest component is computed. The method exploits the fact that the imaginary part of this (complex) ratio closely approximates the frequency of the mode. It also lends itself well to automation and has been tested over a large number of FTMs of order as high as 250.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Vitellins and vitellogenins of Dysdercus koenigii (Heteroptera : Pyrrhocoridae) - identification, purification and temporal pattern

Two vitellins, VtA and VtB, were purified from the eggs of Dysdercus koenigii by gel filtration and ion exchange chromatography. VtA and VtB have molecular weights of 290 and 260 kDa, respectively. Both Vts are glycolipoproteinaceous in nature. VtA is composed of three polypeptides of M-r 116, 92 and 62 kDa while VtB contained an additional subunit of M-r 40 kDa. All subunits except the 116-kDa subunit are glycolipopolypeptides. Polyclonal antibody raised against VtA (anti-VtA antibody) cross-reacted with VtB and also with vitellogenic haemolymph and ovaries and pre-vitellogenic fat bodies, but not with haemolymph from either adult male, fifth instar female, or pre-vitellogenic females demonstrating sex and stage specificity of the Vts. Immunoblots in the presence of anti-VtA revealed two proteins (of 290 and 260 kDa) in both vitellogenic haemolymph and pre-vitellogenic fat bodies that are recognised as D. koenigii Vgs. In newly emerged females, Vgs appeared on day 1 in fat bodies and on day 3 in haemolymph and ovaries. Vg concentration was maximum on day 2 in fat body, day 4 in haemolymph and day 7 in ovary. Although the biochemical and temporal characteristics of these proteins show similarity to some hemipterans, they are strikingly dissimilar with those of a very closely related species. (C) 1999 Elsevier Science Inc. All rights reserved.

Identification of delamination in composite beams using spectral estimation and a genetic algorithm

An efficient strategy for identification of delamination in composite beams and connected structures is presented. A spectral finite-element model consisting of a damaged spectral element is used for model-based prediction of the damaged structural response in the frequency domain. A genetic algorithm (GA) specially tailored for damage identification is derived and is integrated with finite-element code for automation. For best application of the GA, sensitivities of various objective functions with respect to delamination parameters are studied and important conclusions are presented. Model-based simulations of increasing complexity illustrate some of the attractive features of the strategy in terms of accuracy as well as computational cost. This shows the possibility of using such strategies for the development of smart structural health monitoring softwares and systems.

Tool for Identification of Duplicate Records Downloaded from Multiple Cd-Roms. A Case Study with Spirs Based Databases

As research becomes more and more interdisciplinary, literature search from CD-ROM databases is often carried out on more than one CD-ROM database. This results in retrieving duplicate records due to same literature being covered (indexed) in more than one database. The retrieval software does not identify such duplicate records. Three different programs have been written to accomplish the task of identifying the duplicate records. These programs are executed from a shell script to minimize manual intervention. The various fields that have been used (extracted) to identify the duplicate records include the article title, year, volume number, issue number and pagination. The shell script when executed prompts for input file that may contain duplicate records. The programs identify the duplicate records and write them to a new file.

Assessment of genetic diversity and identification of core collection in sandalwood germplasm using RAPDS

Sandalwood is an economically important aromatic tree belonging to the family Santalaceae. The trees are used mainly for their fragrant heartwood and oil that have immense potential for foreign exchange. Very little information is available on the genetic diversity in this species. Hence studies were initiated and genetic diversity estimated using RAPD markers in 51 genotypes of Santalum album procured from different geographcial regions of India and three exotic lines of S. spicatum from Australia. Eleven selected Operon primers (10mer) generated a total of 156 consistent and unambiguous amplification products ranging from 200bp to 4kb. Rare and genotype specific bands were identified which could be effectively used to distinguish the genotypes. Genetic relationships within the genotypes were evaluated by generating a dissimilarity matrix based on Ward's method (Squared Euclidean distance). The phenetic dendrogram and the Principal Component Analysis generated, separated the 51 Indian genotypes from the three Australian lines. The cluster analysis indicated that sandalwood germplasm within India constitutes a broad genetic base with values of genetic dissimilarity ranging from 15 to 91 %. A core collection of 21 selected individuals revealed the same diversity of the entire population. The results show that RAPD analysis is an efficient marker technology for estimating genetic diversity and relatedness, thereby enabling the formulation of appropriate strategies for conservation, germplasm management, and selection of diverse parents for sandalwood improvement programmes.

Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

Identification and characterization of cysteine proteinases of Trypanosoma evansi

Trypanosoma evansi is a causative agent of `surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.

Pruned Universal Symbol Sequences for LZW based Language identification

We present a improved language modeling technique for Lempel-Ziv-Welch (LZW) based LID scheme. The previous approach to LID using LZW algorithm prepares the language pattern table using LZW algorithm. Because of the sequential nature of the LZW algorithm, several language specific patterns of the language were missing in the pattern table. To overcome this, we build a universal pattern table, which contains all patterns of different length. For each language it's corresponding language specific pattern table is constructed by retaining the patterns of the universal table whose frequency of appearance in the training data is above the threshold.This approach reduces the classification score (Compression Ratio [LZW-CR] or the weighted discriminant score[LZW-WDS]) for non native languages and increases the LID performance considerably.