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.

Role of lattice defects and crystallite morphology in the UV and visible-light-induced photo-catalytic properties of combustion-prepared TiO2

The physico-chemical, photo-physical and micro-structural properties responsible for the strikingly different photocatalytic behavior of combustion-prepared TiO2 (c.TiO2) and Degussa P25 (d.TiO2) samples are elucidated in this study. Electron microscopy and selected area electron diffraction micrographs revealed that the two samples exhibited different morphologies. The grains of c.TiO2 were spherical and comprised of 5-6 nm size primary particle. On the other hand, d.TiO2 consisted of large (0.5-3.0 mu m) size and irregular shape aggregates having primary particles of 15-40 nm cross-sectional diameter. The ESR study revealed that the presence of certain defect states in c.TiO2 helped in stabilization of O-. and Ti3+-OH type species during room-temperature UV-irradiation. No such paramagnetic species were however formed over d.TiO2 under similar conditions. C1s and Ti 2p XPS spectra provide evidence for the presence of some lattice vacancies in c.TiO2 and also for the bulk Ti4+ -> Ti3+ conversion during its UV-irradiation. Compared to d.TiO2, c.TiO2 displayed considerably higher activity for discoloration of methyl orange but very poor activity for splitting of water, both under UV and visible light radiations. This is attributed to enhanced surface adsorption of dye molecules over c.TiO2, because of its textural features and also the presence of photo-active ion-radicals. On the other hand, the poor activity of c.TiO2 for water splitting is related to certain defect-induced inter-band charge trapping states in the close vicinity of valence and conduction bands of c.TiO2, as revealed by thermoluminescence spectroscopy. Further, the dispersion of nanosize gold particles gave rise to augmented activity of both the catalysts, particularly for water splitting. This is explained by the promotional role of Au-0 or Au-0/TiO2 interfacial sites in the adsorption and charge-adsorbate interaction processes. (C) 2011 Elsevier B.V. All rights reserved.

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.

Synthesis and characterization of photo-crosslinkable main-chain liquid-crystalline polymers containing bis(benzylidene)cycloalkanone units

A series of new photo-crosslinkable main-chain liquid-crystalline polymers containing bis(benzylidene)cycloalkanone units have been studied. These units in the polymers function as mesogens as well as photoactive centres. Polyesters with three different bis(4-hydroxybenzylidene)cycloalkanones corresponding to three cycloalkanones, namely cyclopentanone, cyclohexanone and cycloheptanone, have been prepared. Three dicarboxylic acids with ether linkages, which were derived from oligoethylene oxides, namely triethylene glycol, tetraethylene glycol and pentaethylene glycol, have been used as spacers in these polymers. Polymerization was carried out by both solution and interfacial polycondensation; the latter method gave high-molecular-weight polymers. Structural characterizations were done by ultra-violet, infra-red and H-1 nuclear magnetic resonance spectroscopy. Liquid-crystalline properties were studied by differential scanning calorimetry and polarized-light optical microscopy. These polymers show a nematic mesophase. Liquid-crystalline transition temperatures were correlated with polymer structure. The decrease in transition temperature with increase in cycloalkanone ring size was explained in terms of the change in geometrical anisotropy of bis(benzylidene)cycloalkanone units. MNDO (modified neglect of differential overlap) calculations were performed on the model compounds, bis(4-acetyloxybenzylidene)cycloalkanone to elucidate the geometrical variation of the mesogenic units with cycloalkanone ring size. Studies of photolysis reveal the two kinds of photoreactions that proceed in these polymer systems, namely photoisomerization and photo-crosslinking. The former reaction disrupts the parallel stacking of the chromophores and is reflected as an increase in the ultra-violet spectral intensity. The favourability of these two reactions depends on the mobility of the polymer chains. When the photolysis was done below T-g, photo-crosslinking dominates over photoisomerization. Above T-g, photoisomerization is followed by photo-crosslinking. The photosensitivity of the polymers decreases with increase in size of the cycloalkanone ring.

Novel photo-crosslinkable flame retardant polyvanillylidene arylphosphate esters

A new class of photo-crosslinkable flame retardant arylphosphate ester polymers based on diarylidenecycloalkanone groups has been synthesized by polymerizing 2,5-divanillylidene cyclopentanone and 2,6-divanillylidenecyclohexanone with various arylphosphorodichloridates by interfacial polycondensation using a phase transfer catalyst. The resulting polymers were characterized by inherent viscosity, g.p.c., i.r., H-1, C-13, P-31 n.m.r. spectroscopy. These polymers were studied for their photochemical and flame retardant properties. The divanillylidene cycloalkanone group in the chain function as photoactive centres while arylphosphate ester groups impart flame retardancy. The photo-crosslinking proceeds via 2 pi + 2 pi cycloaddition reaction of the divanillylidene cycloalkanone moieties. The crosslinking rate, thermal stability and flammability characteristics of the polymers increase with decrease in the size of the cycloalkanone ring. (C) 1997 Elsevier Science Ltd.

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.

Photo-Defined Electrically Assisted Etching Method for Metal Stencil Fabrication

Metal stencils are well known in electronics printing application such as for dispensing solder paste for surface mounting, printing embedded passive elements in multilayer structures, etc. For microprinting applications using stencils, the print quality depends on the smoothness of the stencil aperture and its dimensional accuracy, which in turn are invariably related to the method used to manufacture the stencils. In this paper, fabrication of metal stencils using a photo-defined electrically assisted etching method is described. Apertures in the stencil were made in neutral electrolyte using three different types of impressed current, namely, dc, pulsed dc, and periodic pulse reverse (PPR). Dimensional accuracy and wall smoothness of the etched apertures in each of the current waveforms were compared. Finally, paste transfer efficiency of the stencil obtained using PPR was calculated and compared with those of a laser-cut electropolished stencil. It is observed that the stencil fabricated using current in PPR waveform has better dimensional accuracy and aperture wall smoothness than those obtained with dc and pulsed dc. From the paste transfer efficiency experiment, it is concluded that photo-defined electrically assisted etching method can provide an alternate route for fabrication of metal stencils for future microelectronics printing applications.