Synthesis and supramolecular assembly of the bifunctional borinic acid 1,2-fcB(OH)](2)

Treatment of the chloro-substituted diboradiferrocene derivative 1 with Me3SiOMe and subsequent hydrolysis resulted in formation of the novel organometallic bis(borinic acid) derivative 3. The assembly of 3 into supramolecular structures via hydrogen bonding and reversible covalent boron-oxygen bond formation was explored. Upon crystallization from acetone or THF one-dimensional chains form in which molecules of 3 alternately serve as hydrogen bond donors and acceptors. The additional OH hydrogens that are not involved in hydrogen bonding within the polymeric chains undergo hydrogen bonding to the solvent molecules. Removal of the solvent was achieved at moderate temperature under high vacuum. While the polymeric chains remain intact, in the absence of the solvent as a hydrogen bond acceptor, short contacts to the Cp rings of neighboring polymer strands lead to a network-like structure. At higher temperatures, further dehydration occurs with formation of B-O-B linkages as confirmed by MALDI-TOF mass spectrometry. Oligomers with up to 15 repeating units (30 ferrocenes) were detected.

Plasmodium falciparum Prp16 homologue and its role in splicing

Large numbers of Plasmodium genes have been predicted to have introns. However, little information exists on the splicing mechanisms in this organism. Here, we describe the DExD/DExH-box containing Pre-mRNA processing proteins (Prps), PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p, present in the Plasmodium falciparum genome and characterized the role of one of these factors, PfPrp16p. It is a member of DEAH-box protein family with nine collinear sequence motifs, a characteristic of helicase proteins. Experiments with the recombinantly expressed and purified PfPrp16 helicase domain revealed binding to RNA, hydrolysis of ATP as well as catalytic helicase activities. Expression of helicase domain with the C-terminal helicase-associated domain (HA2) reduced these activities considerably, indicating that the helicase-associated domain may regulate the PfPrp16 function. Localization studies with the PfPrp16 GFP transgenic lines suggested a role of its N-terminal domain (1-80 amino acids) in nuclear targeting. Immunodepletion of PfPrp16p, from nuclear extracts of parasite cultures, blocked the second catalytic step of an in vitro constituted splicing reaction suggesting a role for PfPrp16p in splicing catalysis. Further we show by complementation assay in yeast that a chimeric yeast-Plasmodium Prp16 protein, not the full length PfPrp16, can rescue the yeast prp16 temperature-sensitive mutant. These results suggest that although the role of Prp16p in catalytic step II is highly conserved among Plasmodium, human and yeast, subtle differences exist with regards to its associated factors or its assembly with spliceosomes. (C) 2012 Elsevier B.V. All rights reserved.

Synthesis, characterization and phosphotriesterase mimetic activity of some Zn(II) and Cu(II) complexes

We report here the synthesis and characterization of a few phenolate-based ligands bearing tert- amino substituent and their Zn(II) and Cu(II) metal complexes. Three mono/binuclear Zn(II) and Cu(II) complexes Zn(L1)(H2O)].CH3OH.H2O (1) (H (2) L1 = 6,6(')-(((2-dimethylamino)ethylazanediyl)bis(methylene))bis(2, 4-dimethylphenol), Zn-2(L2)(2)] (2) (H (2) L2 = 2,2(')-(((2-dimethylamino)ethyl)azanediyl)bis(methylene)bis(4- methylphenol) and Cu-2(L3)(2).CH2 Cl-2] (3) (H (2) L3 = (6,6(')-(((2-(diethylamino)ethyl)azanediyl)bis(methylene)) bis(methylene))bis(2,4-dimethylphenol) were synthesized by using three symmetrical tetradendate ligands containing N2O2 donor sites. These complexes are characterized by a variety of techniques including; elemental analysis, mass spectrometry, H-1, C-13 NMR spectroscopic and single crystal X-ray analysis. The new complexes have been tested for the phosphotriesterase (PTE) activity with the help of P-31 NMR spectroscopy. The P-31 NMR studies show that mononuclear complex Zn(L1)(H2O)].CH3OH.H2O (1) can hydrolyse the phosphotriester i.e., p-nitrophenyl diphenylphosphate (PNPDPP), more efficiently than the binuclear complexes Zn-2(L2)(2)] (2) and Cu-2(L3)(2).CH2Cl2] (3). The mononuclear Zn(II) complex (1) having one coordinated water molecule exhibits significant PTE activity which may be due to the generation of a Zn(II)-bound hydroxide ion during the hydrolysis reactions in CHES buffer at pH 9.0.

Biochemical characterization of C4 protein of cotton leaf curl Kokhran virus-Dabawali

Background: Cotton leaf curl Kokhran Virus-Dabawali (CLCuKV-Dab) is a monopartite begomovirus encoding two proteins V1 and V2 in the virion sense and four proteins Cl, C2, C3 and C4 in the complementary sense. The C4 protein of monopartite begomoviruses has been implicated to play a role in symptom determination and virus movement. The present work aims at the biochemical characterization of this protein. Methods: The C4 protein of CLCuKV-Dab was purified in fusion with GST and tested for the ability to hydrolyze ATP and other phosphate containing compounds. ATPase activity was assayed by using radiolabeled gamma-32P]-ATP and separating the product of reaction by thin layer chromatography. The hydrolysis of other compounds was monitored by the formation of a blue colored phosphomolybdate complex which was estimated by measuring the absorbance at 655 nm. Results: The purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Deletion of a sequence resembling the catalytic motif present in phosphotyrosine phosphatases resulted in 70% reduction in both the activities. Mutational analysis suggested arginine 13 to be catalytically important for the ATPase and cysteine 8 for the pyrophosphatase activity of GST-C4. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4. Conclusions: The residues important for the enzymatic activities of GST-C4 are present in a motif different from the classical Walker motifs and the non-classical ATP binding motifs reported so far. General significance: The C4 protein of CLCuKV-Dab, a putative natively unfolded protein, exhibits enzymatic activities.

Kinetic proofreading at single molecular level: aminoacylation of tRNA(Ile) and the role of water as an editor

Proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. In this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. We consider aminoacylation of tRNA(Ile) which is a crucial step in protein synthesis and for which experimental results are now available. We present an augmented kinetic scheme and then employ methods of stochastic simulation algorithm to obtain time dependent concentrations of different substances involved in the reaction and their rates of formation. We obtain the rates of product formation and ATP hydrolysis for both correct and wrong substrates (isoleucine and valine in our case, respectively), in single molecular enzyme as well as ensemble enzyme kinetics. The present theoretical scheme correctly reproduces (i) the amplitude of the discrimination factor in the overall rates between isoleucine and valine which is obtained as (1.8x10(2)).(4.33x10(2)) = 7.8x10(4), (ii) the rates of ATP hydrolysis for both Ile and Val at different substrate concentrations in the aminoacylation of tRNA(Ile). The present study shows a non-michaelis type dependence of rate of reaction on tRNA(Ile) concentration in case of valine. The overall editing in steady state is found to be independent of amino acid concentration. Interestingly, the computed ATP hydrolysis rate for valine at high substrate concentration is same as the rate of formation of Ile-tRNA(Ile) whereas at intermediate substrate concentration the ATP hydrolysis rate is relatively low. We find that the presence of additional editing domain in class I editing enzyme makes the kinetic proofreading more efficient through enhanced hydrolysis of wrong product at the editing CP1 domain.

DNA and protein targeting 1,2,4-triazole based water soluble dinickel(II) complexes enhances antiproliferation and lactate dehydrogenase inhibition

Water soluble dinickel(II) complexes Ni-2(L)(2)(1-2)](NO3)(4) (1-2), where L1-2 are triazole based dinucleating ligands, were synthesized and characterized. The DNA binding, protein binding, DNA hydrolysis and anticancer properties were investigated. The interactions of complexes 1 and 2 with calf thymus DNA were studied by spectroscopic techniques, including absorption and fluorescence spectroscopy. The DNA binding constant values of the complexes 1 and 2 were found to be 2.36 x 10(5) and 4.87 x 10(5) M-1 and the binding affinities are in the following order: 2 > 1. Both the dinickel(II) complexes 1 and 2, promoted the hydrolytic cleavage of plasmid pBR322 DNA under both anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 1 and 2 under physiological conditions give the observed rate constants (k(obs)) of 5.05 +/- 0.2 and 5.65 +/- 0.1 h(-1), respectively, which shows 10(8)-fold rate acceleration over the uncatalyzed reaction of ds-DNA. Meanwhile, the interactions of the complex with BSA have also been studied by spectroscopy. Both the complexes 1 and 2 display strong binding propensity and the binding constant (K-b), number of binding sites (n) were obtained are 0.71 x 10(6) 1.47] and 5.62 x 10(6) 1.98] M-1, respectively. The complexes 1 and 2 also promoted the apoptosis against human carcinoma (HeLa, and BeWo) cancer cells. Cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme level in cancer cell lysate and content media. (c) 2013 Elsevier Ltd. All rights reserved.

The design of polyaniline based sensor for the qualitative estimation of malonaldehyde

The detection of contaminated food in every stage of processing required new technology for fast identification and isolation of toxicity in food. Since effect of food contaminant are severe to human health, the need of pioneer technologies also increasing over last few decades. In the current study, MDA was prepared by hydrolysis of 1,1,3,3-tetramethoxypropane in HCl media and used in the electrochemical studies. The electrochemical sensor was fabricated with modified glassy carbon electrode with polyaniline. These sensors were used for detection of sodium salt of malonaldehyde and observed that a high sensitivity in the concentration range similar to 1 x 10(-1) M and 1 x 10(-2) M. Tafel plots show the variation of over potential from -1.73 V to -3.74 V up to 10(-5) mol/L indicating the lower limit of detection of the system. (C) 2013 Elsevier Ltd. All rights reserved.

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

Rapid determination of main constituents of packed juices by reverse phase-high performance liquid chromatography: an insight in to commercial fruit drinks

The present work reports the compositional analysis of thirteen different packed fruit juices using high performance liquid chromatography (HPLC). Vitamin C, organic acids (citric and malic) and sugars (fructose, glucose and sucrose) were separated, analyzed and quantified using different reverse phase methods. A new rapid reverse phase HPLC method was developed for routine analysis of vitamin C in fruit juices. The precision results of the methods showed that the relative standard deviations of the repeatability and reproducibility were < 0.05 and < 0.1 respectively. Correlation coefficient of the calibration models developed was found to be higher than 0.99 in each case. It has been found that the content of Vitamin C was less variable amongst different varieties involved in the study. It is also observed that in comparison to fresh juices, the packed juices contain lesser amounts of vitamin C. Citric acid was found as the major organic acids present in packed juices while maximum portion of sugars was of sucrose. Comparison of the amount of vitamin C, organic acids and sugars in same fruit juice of different commercial brands is also reported.

Facile hydrothermal synthesis of carbon nanoparticles and possible application as white light phosphors and catalysts for the reduction of nitrophenol

We report a simple hydrothermal synthesis of highly reproducible carbon nanoparticles in a size range between 2 and 7 nmfroma single precursor sucrose without either surface passivating agents or acids and bases. The carbon nanoparticles can be used as white light phosphors, especially for ultraviolet light emitting diodes and metal-free catalyst for the reduction of nitrophenol.

Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila

Cytosolic nucleotidase II (cN-II) from Legionellapneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation. DatabaseThe coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers and . The accession number of GMP complexed LpcN-II is . Structured digital abstract andby() andby() Structured digital abstract was added on 5 March 2014 after original online publication]

The knight of sugar industry: T. S. Venkatraman (1884-1963)

Tiruvadi Sambasiva Venkatraman (TSV) was a plant breeder. In response to a call from Pundit Madan Mohan Malaviya, he made it his mission to develop high-yielding varieties of sugarcane for manufacturing sugar and making it available as a sweetening agent and an energy source for the malnourished children of India. Using Saccharum officinarum, then under cultivation in India, as the female parent, he artificially fertilized it with pollen from S. barberi, which grew wild in Coimbatore. After 4-5 recurrent backcrossings of S. officinarum Chi wild Sorghum spontaneum with S. officinarum as the female parent, TSV selected the `rare' interspecies hybrid cane varieties that resembled sugarcane and had approximately 2.5 cm thick juicy stems containing 16-18% sucrose - nearly 35 times more than what occurred in parent stocks. The hybrid canes matured quickly, were resistant to waterlogging, drought, and to the red-rot disease caused by Glomerella tucumanensis (Sordariomycetes: Glomerellaceae), and to the sereh-virus disease. Most importantly, they were amenable for propagation using stem cuttings. In recognition of the development of high-yielding sugarcane varieties, TSV was conferred the titles Rao Bahadur, Rao Sahib, and Sir by the British Government, and Padma Bhushan by the Republic of India. In the next few decades, consequent to TSV's work, India turned into the second largest sugar producer in the world, after Brazil. The hybrid sugarcane varieties developed are the foundational stocks for new sugarcane x bamboo hybrids, and for possible resistance to Puccinia megalocephala (Pucciniomycetes: Pucciniaceae) and Ustilago scitaminea (Ustilaginomycetes: Ustilaginaceae) using molecular techniques.

In vitro exposure of tobacco specific nitrosamines decreases the rat lung phospholipids by enhanced phospholipase A(2) activity

Tobacco-specific nitrosamines (TSNA) have implications in the pathogenesis of various lung diseases and conditions are prevalent even in non-smokers. N-nitrosonornicotine (NNN) and 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are potent pulmonary carcinogens present in tobacco product and are mainly responsible for lung cancer. TSNA reacts with pulmonary surfactants, and alters the surfactant phospholipid. The present study was undertaken to investigate the in vitro exposure of rat lung tissue slices to NNK or NNN and to monitor the phospholipid alteration by P-32]orthophosphate labeling. Phospholipid content decreased significantly in the presence of either NNK or NNN with concentration and time dependent manner. Phosphatidylcholine (PC) is the main phospholipid of lung and significant reduction was observed in PC similar to 61%, followed by phosphatidylglycerol (PG) with 100 mu M of NNK, whereas NNN treated tissues showed a reduction in phosphatidylserine (PS) similar to 60% and PC at 250 mu M concentration. The phospholipase A(2) assays and expression studies reveal that both compounds enhanced phospholipid hydrolysis, thereby reducing the phospholipid content. Collectively, our data demonstrated that both NNK and NNN significantly influenced the surfactant phospholipid level by enhanced phospholipase A(2) activity. (C) 2014 Elsevier Ltd. All rights reserved.

The Non-catalytic ``Cap Domain'' of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Despite highly conserved core catalytic domains, members of the metallophosphoesterase (MPE) superfamily perform diverse and crucial functions ranging from nucleotide and nucleic acid metabolism to phospholipid hydrolysis. Unique structural elements outside of the catalytic core called ``cap domains'' are thought to provide specialization to these enzymes; however, no directed study has been performed to substantiate this. The cap domain of Rv0805, an MPE from Mycobacterium tuberculosis, is located C-terminal to its catalytic domain and is dispensable for the catalytic activity of this enzyme in vitro. We show here that this C-terminal extension (CTE) mediates in vivo localization of the protein to the cell membrane and cell wall as well as modulates expression levels of Rv0805 in mycobacteria. We also demonstrate that Rv0805 interacts with the cell wall of mycobacteria, possibly with the mycolyl-arabinogalactan-peptidoglycan complex, by virtue of its C terminus, a hitherto unknown property of this MPE. Using a panel of mutant proteins, we identify interactions between active site residues of Rv0805 and the CTE that determine its association with the cell wall. Finally, we show that Rv0805 and a truncated mutant devoid of the CTE produce different phenotypic effects when expressed in mycobacteria. Our study thus provides a detailed dissection of the functions of the cap domain of an MPE and suggests that the repertoire of cellular functions of MPEs cannot be understood without exploring the modulatory effects of these subdomains.

Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis

We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.