Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Identification of carbohydrate structures as receptors for localised adherent enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli strains of diffused adherent (DA) and localised adherent (LA) phenotypes were tested for their ability to bind to glycolipids. DA strains did not bind to the glycolipids tested, while LA strains bound to asialo GM1, asialo GM2, globoside and lacto-N-neotetraose in decreasing order of avidity. The minimum common sequence among the four glycolipids could be delineated as GalNac β 1–4 Gal as the binding epitope with GalNac β 1–3 Gal and GlcNac β 1–3 Gal serving as relatively weaker binders. The binding was not inhibited by a variety of free oligosaccharides or by the neoglycoproteins tested. Adhesion-negative mutants of an enteropathogenic LA strain showed a markedly reduced binding to asialo GM1 indicating that the recognition of GalNac β 1–4 Gal was correlated with the ability to adhere to HeLa cells. Thus recognition and binding to glycolipids could play an important role in colonisation through adherence to intestinal surfaces.

Applicability of partition ratios, mercuric chloride complexes, and chromatographic behavior for the identification of carotenoids

Partition ratios and M50 values of different carotenoids in hexaneaqueous methanol were determined. Mercuric chloride complexes of 14 epoxy carotenoids were prepared and their absorption maxima in acetone were estimated. The difference in chromatographic behavior of carotenoid epoxides on alumina and magnesium oxide-Celite columns is discussed. It is shown that the magnesium oxide-Celite column behaves as a reverse-phase chromatographic column to alumina column.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3

The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 95.62, c = 149.13 angstrom. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V-M) of 2.3 angstrom(3) Da(-1), corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an similar to 60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix ( bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors.

Association of methylenetetrahydrofolate reductase gene polymorphisms & colorectal cancer in India

Background & objectives: Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and involved in DNA synthesis, DNA repair and DNA methylation. The two common functional polymorphisms of MTHFR, 677C -> T and 1298 A -> C have shown to impact several diseases including cancer. This case-control study was undertaken to analyse the association of the MTHFR gene polymorphisms 677 C -> T and 1298 A -> C and risk of colorectal cancer (CRC).Methods: One hundred patients with a confirmed histopathologic diagnosis of CRC and 86 age and gender matched controls with no history of cancer were taken for this study. DNA was isolated from peripheral blood samples and the genotypes were determined by PCR-RFLP. The risk association was estimated by compounding odds ratio (OR) with 95 per cent confidence interval (CI). Results: Genotype frequency of MTHFR 677 CC, CT and TT were 76.7, 22.1 and 1.16 per cent in controls, and 74,25 and 1.0 per cent among patients. The 'T' allele frequency was 12.21 and 13.5 per cent in controls and patients respectively. The genotype frequency of MTHFR 1298 AA, AC, and CC were 25.6, 58.1 and 16.3 per cent for controls and 22, 70 and 8 per cent for patents respectively. The 'C' allele frequency for 1298 A -> C was 43.0 and 45.3 per cent respectively for controls and patients. The OR for 677 CT was 1.18 (95% CI 0.59-2.32, P = 0.642), OR for 1298 AC was 1.68 (95% CI 0.92-3.08, P = 0.092) and OR for 1298 CC was 0.45(95% CI 0.18-1.12, P = 0.081). The OR for the combined heterozygous state (677 CT and 1298 AC) was 1.18(95% CI 0.52-2.64, P =0.697).Interpretation & conclusion: The frequency of the MTHFR 677 TT genotype is rare as compared to 1298 CC genotype in the population studied. There was no association between 677 C -> T and 1298 A -> C polymorphisms and risk of CRC either individually or in combination. The homozygous state for 1298 A -> C polymorphism appears to slightly lower risk of CRC. This needs to be confirmed with a larger sample size.

Synthesis and identification of a new class of (S)-2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole derivatives as potent antileukemic agents

Benzothiazoles are multitarget agents with broad spectrum of biological activity. Among the antitumor agents discovered in recent years, the identification of various 2-(4-aminophenyl) benzothiazoles as potent and selective antitumor drugs against different cancer cell lines has stimulated remarkable interest. Some of the benzothiazoles are known to induce cell cycle arrest, activation of caspases and interaction with DNA molecule. Based on these interesting properties of benzothiazoles and to obtain new biologically active agents, a series of novel 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5(a-i) were synthesized and evaluated for their efficacy as antileukemic agents in human leukemia cells (K562 and Reh). The chemical structures of the synthesized compounds were confirmed by H-1 NMR, LCMS and IR analysis. The cytotoxicity of these compounds were determined using trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results showed that, these compounds mediate a significant cytotoxic response to cancer cell lines tested. We found that the compounds having electron withdrawing groups at different positions of the phenyl ring of the thiourea moiety displayed significant cytotoxic effect with IC50 value less than 60 mu M. To rationalize the role of electron withdrawing group in the induction of cytotoxicity, we have chosen molecule 5g (IC50 similar to 15 mu M) which is having chloro substitution at ortho and para positions. Flow cytometric analysis of annexin V-FITC/ propidium iodide (PI) double staining and DNA fragmentation suggest that 5g can induce apoptosis.

Enzyme catalysed non-oxidative decarboxylation of aromatic acids II. Identification of active site residues of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger

In order to understand the mechanism of decarboxylation by 2,3-dihydroxybenzoic acid decarboxylase, chemical modification studies were carried out. Specific modification of the amino acid residues with diethylpyrocarbonate, N-bromosuccinimide and N-ethylmaleiimide revealed that at least one residue each of histidine, tryptophan and cysteine were essential for the activity. Various substrate analogs which were potential inhibitors significantly protected the enzyme against inactivation. The modification of residues at low concentration of the reagents and the protection experiments suggested that these amino acid residues might be present at the active site. Studies also suggested that the carboxyl and ortho-hydroxyl groups of the substrate are essential for interaction with the enzyme.

An intelligent approach using support vector machines for monitoring and identification of faults on transmission systems

Power system disturbances are often caused by faults on transmission lines. When faults occur in a power system, the protective relays detect the fault and initiate tripping of appropriate circuit breakers, which isolate the affected part from the rest of the power system. Generally Extra High Voltage (EHV) transmission substations in power systems are connected with multiple transmission lines to neighboring substations. In some cases mal-operation of relays can happen under varying operating conditions, because of inappropriate coordination of relay settings. Due to these actions the power system margins for contingencies are decreasing. Hence, power system protective relaying reliability becomes increasingly important. In this paper an approach is presented using Support Vector Machine (SVM) as an intelligent tool for identifying the faulted line that is emanating from a substation and finding the distance from the substation. Results on 24-bus equivalent EHV system, part of Indian southern grid, are presented for illustration purpose. This approach is particularly important to avoid mal-operation of relays following a disturbance in the neighboring line connected to the same substation and assuring secure operation of the power systems.

Identification of corona discharge-induced SF6 oxidation mechanisms using SF6/18O2/H216O and SF6/16O2/H218O gas mixtures

The absolute yields of gaseous oxyfluorides SOF2, SO2F2, and SOF4 from negative, point-plane corona discharges in pressurized gas mixtures of SF6 with O2 and H2O enriched with18O2 and H2 18O have been measured using a gas chromatograph-mass spectrometer. The predominant SF6 oxidation mechanisms have been revealed from a determination of the relative18O and16O isotope content of the observed oxyfluoride by-product. The results are consistent with previously proposed production mechanisms and indicate that SOF2 and SO2F2 derive oxygen predominantly from H2O and O2, respectively, in slow, gas-phase reactions involving SF4, SF3, and SF2 that occur outside of the discharge region. The species SOF4 derives oxygen from both H2O and O2 through fast reactions in the active discharge region involving free radicals or ions such as OH and O, with SF5 and SF4.

Crystallographic identification of a ‘stepped-cubane’ structure for the Cu4O4 core in [Cu4L2(bipy)4(µ3-OH)2][ClO4]4(HL = 5-hydroxy-6-methylpyridine-,4-dimethanol, bipy = 2,2′-bipyridine)

The structure of [Cu4L2(bipy)4(µ3-OH)2][ClO4]4 containing a Vitamin B6 ligand, pyridoxine (5-hydroxy-6-methylpyridine-3,4-dimethanol, HL), and 2,2′-bipyridine (bipy) has been determined by single-crystal X-ray analysis. This is the first report on a copper(II) complex having a ‘stepped-cubane’ structure. The compound crystallizes in the triclinic space group P[1 with combining macron](Z= 1) with a= 11.015(3), b= 11.902(1), c= 13.142(2)Å, α= 105.07(1), β= 102.22(1) and γ= 99.12(1)°; R= 0.054). The co-ordination geometry around each copper is trigonally distorted square pyramidal. Two of the basal sites are occupied by bipyridyl nitrogens in a bidentate fashion. The remaining basal positions for Cu(1) are filled by a phenolic oxygen and a 4-hydroxymethyl oxygen of the L moiety, whereas for Cu(2) they are occupied by two µ3-OH oxygens. The axial sites are occupied by a µ3-OH oxygen and the 4-hydroxymethyl oxygen of the same pyridoxine for Cu(1) and Cu(2), respectively. Both the bridging nature of the 4-hydroxymethyl oxygen of the L moiety and the unsymmetrical bridging nature of the µ3-OH groups with axial–equatorial bridging are novel features. The structure is discussed in relation to stepped-cubane structures reported in the literature. A comparative study is also made with µ3-hydroxo-bridged copper(II) complexes. Both the plasticity effect of CuII and the stacking interactions between the various rings appear to be important in stabilizing this unusual structure.

Involvement of heme in the transcriptional activation of CYPIIB1/B2 gene by phenobarbitone in rat liver—Studies with succinylacetone

Earlier studies in this laboratory had implicated heme to function as a positive modulator of phenobarbitonemediated activation of CYPIIB1/B2 gene transcription in rat liver. However, recent reports have indicated that succinylacetone, a specific inhibitor of δ-aminolevulinate dehydrase, does not affect this process. The present studies indicate that succinylacetone does inhibit the phenobarbitone-mediated increase in CYPIIB1/B2 mRNAs and their transcription in rat liver at early time points (45 min to 3 h), but the inhibition is not pronounced at later time points (16 h). Succinylacetone is a weaker inhibitor of heme biosynthesis than CoCl2, 3-amino-1,2,4-triazole, or thioacetamide used earlier in this laboratory. Succinylacetone induces δ-aminolevulinate synthase, whereas the other compounds depress the levels of the enzyme. There is a good correlation between the amount of freshly synthesized nuclear heme pool and the activation of CYPIIB1/B2 transcription by phenobarbitone. A model implicating a nuclear heme pool regulating the transcription of δ-aminolevulinate synthase, CYPIIB1/ B2, and heme oxygenase genes is proposed.

The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) angstrom and a = 6.023(3) angstrom, b = 11.719(3) angstrom and b = 13.455(4) angstrom, c = 20.409(3) angstrom and c = 20.702(4) angstrom for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.

Nuclear-quadrupole resonance in coordination chemistry- identification of bridiging and therminal chlorines in the mercuric chloride-4-picoline n-oxide complex

The temperature dependence of the chlorine-35 n.q.r. in the mercuric chloride-4-picoline N-oxide complex has been studied from 77 K to room temperature, and the results are used to assign the observed frequencies to terminal and bridging chlorines.