Identifying N60D mutation in omega subunit of Escherichia coli RNA polymerase by bottom-up proteomic approach

Escherichia coli RNA polymerase is a multi-subunit enzyme containing alpha(2)beta beta'omega sigma, which transcribes DNA template to intermediate RNA product in a sequence specific manner. Although most of the subunits are essential for its function, the smallest subunit omega (average molecular mass similar to 10,105 Da) can be deleted without affecting bacterial growth. Creating a mutant of the omega subunit can aid in improving the understanding of its role. Sequencing of rpoZ gene that codes for omega subunit from a mutant variant suggested a substitution mutation at position 60 of the protein: asparagine (N) -> aspartic acid (D). This mutation was verified at the protein level by following a typical mass spectrometry (MS) based bottom-up proteomic approach. Characterization of in-gel trypsin digested samples by reverse phase liquid chromatography (LC) coupled to electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) enabled in ascertaining this mutation. Electron transfer dissociation (ETD) of triply charged (M + 3H)(3+)] tryptic peptides (residues 53-67]), EIEEGLINNQILDVR from wild-type and EIEEGLIDNQILDVR from mutant, facilitated in unambiguously determining the site of mutation at residue 60.

Identification and characterization of a library of microheterogeneous cyclohexa-depsipeptides from the fungus

Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a beta-hydroxy acid residue and five alpha-amino acids, while isaridins contain a beta-amino acid, an alpha-hydroxy acid, and four alpha-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H](+) ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid alpha beta C-11 turn, formed by the beta-hydroxy acid and glycine residues and a (D)Leu-(L)Ala type II' beta-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.

Studies on simultaneous nhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences.The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.

Src Homology 3-interacting Domain of Rv1917c of Mycobacterium tuberculosis Induces Selective Maturation of Human Dendritic Cells by Regulating PI3K-MAPK-NF-kappa B Signaling and Drives Th2 Immune Responses

Mycobacterium tuberculosis, an etiological agent of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Pathogenic mycobacteria survive in the host by subverting host innate immunity. Dendritic cells (DCs) are professional antigen-presenting cells that are vital for eliciting immune responses to infectious agents, including pathogenic mycobacteria. DCs orchestrate distinct Th responses based on the signals they receive. In this perspective, deciphering the interactions of the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family of proteins of M. tuberculosis with DCs assumes significant pathophysiological attributes. In this study, we demonstrate that Rv1917c (PPE34), a representative member of the proline-proline-glutamic-major polymorphic tandem repeat family, interacts with TLR2 and triggers functional maturation of human DCs. Signaling perturbations implicated a critical role for integrated cross-talk among PI3K-MAPK and NF-kappa B signaling cascades in Rv1917c-induced maturation of DCs. However, this maturation of DCs was associated with a secretion of high amounts of anti-inflammatory cytokine IL-10, whereas Th1-polarizing cytokine IL-12 was not induced. Consistent with these results, Rv1917c-matured DCs favored secretion of IL-4, IL-5, and IL-10 from CD4(+) T cells and contributed to Th2-skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, the Rv1917c-skewed Th2 immune response involved induced expression of cyclooxygenase-2 (COX-2) in DCs. Taken together, these results indicate that Rv1917c facilitates a shift in the ensuing immunity toward the Th2 phenotype and could aid in immune evasion by mycobacteria.

Detection and characterisation of circulating cysticercal antigens in human cerebrospinal fluid

With biotin labelled and unlabelled immunoglobulin fraction of anticysticercal antibodies raised in rabbits, tandem-enzyme linked immunosorbent assay (T-ELISA), capture-dot immunobinding assay (C-DIA) and reverse passive haemagglutination (RPHA) tests were developed for the detection of cysticercal antigens. The sensitivity levels were respectively, 9 ng ml−1, 2 ng ml−1 and 45 ng ml−1. All three methods were of equal specificity as none of the antigens of Mycobacterium tuberculosis, Japanese encephalitis virus and Echinococcus granulosus reacted with anticysticercal IgG. Cysticercal antigens were detected in the cerebrospinal fluid (CSF) of confirmed neurocysticercosis at sensitivity levels of 91·6% by T-ELISA, 83·33% by C-DIA and 75% by RPHA and specificity levels of >93%. Western analysis of these antigens in CSF showed mainly antigens of 64–68 kDa and 24–28 kDA. By crossed immunoelectrophoresis (CIE) with an intermediate gel technique, five circulating antigens were found to be released from scolex and fluid.

Structure and stability of human telomeric sequence

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)(n). We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T(2)AG(3))(4), d(G(3)T(2)A)(3)G(3), and d(G(3)T(2)AG(3)) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G(3)T(2)A)(3)G(3) and d(T(2)AG(3))(4) assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G(3)T(2)AG(3)) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide D(G(3)T(2)AG(3)) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T(2)AG(3))(4) suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.

Synthesis based on cyclohexadienes .24. A new total synthesis of pupukean-2-one and a facile entry to copa and ylanga type sesquiterpene skeletons

A novel tandem 5-exo-trig allyl and 3-exo-trig radical cyclisation and rearrangement to copa and ylanga type sesquiterpene skeletons from easily prepared cyclohexadienes is reported, A new total synthesis of pupukean-2-one 8, which belongs to a novel class of sesquiterpenes, involving a 5-exo-trig allyl radical cyclisation as the key step is also reported.

``Clickable'', Trifunctional Magnetite Nanoparticles and Their Chemoselective Biofunctionalization

A multifunctional iron oxide based nanoformulation for combined cancer-targeted therapy and multimodal imaging has been meticulously designed and synthesized using a chemoselective ligation approach. Novel superparamagnetic magnetite nanoparticles simultaneously functionalized with amine, carboxyl, and azide groups were fabricated through a sequence of stoichiometrically controllable partial succinylation and Cu (II) catalyzed diazo transfer on the reactive amine termini of 2-aminoethylphosphonate grafted magnetite nanoparticles (MNPs). Functional moieties associated with MNP surface were chemoselectively conjugated with rhodamine B isothiocyanate (RITC), propargyl folate (FA), and paclitaxel (PTX) via tandem nucleophic addition of amine to isothithiocyanates, Cu (I) catalyzed azide-alkyne click chemistry and carbodiimide-promoted esterification. An extensive in vitro study established that the bioactives chemoselectively appended to the magnetite core bequeathed multifunctionality to the nanoparticles without any loss of activity of the functional molecules. Multifunctional nanoparticles, developed in the course of the study, could selectively target and induce apoptosis to folate-receptor (FR) overexpressing cancer cells with enhanced efficacy as compared to the free drug. In addition, the dual optical and magnetic properties of the synthesized nanoparticles aided in the real-time tracking of their intracellular pathways also as apoptotic events through dual fluorescence and MR-based imaging.

Chemistry of tetrathiomolybdate: Applications in organic synthesis

The utility of tetrathiomolybdate in a variety of organic transformations is presented in this account. The sulfur transfer ability of tetrathiomolybdate is exploited in the synthesis of organic disulfides under mild reaction conditions. The induced internal redox reactions associated with tetrathiomolybdate have been thoroughly exploited in developing various methodologies, which include the reduction of organic azides, synthesis of diselenides, cyclic imines, thioamides, and thiolactams. In addition, novel deprotection strategies using tetrathiomolybdate have been developed to cleave the propargyl and propargyloxy carbonyl (POC) protecting groups. Tetrathiomolybdate mediated tandem sulfur transfer-reduction-Michael reactions have been applied to the synthesis of sulfur containing bicyclic systems. Furthermore, the reactions in the solid state and the reactions in water medium assisted by tetrathiomolybdate have greatly simplified the synthesis of organic disulfides.

A facile regioselective construction of spiro epoxy-bridged tetrahydropyranone frameworks

Investigations on the reactivity profile of the transient five-membered-ring cyclic carbonyl ylides, generated from alpha-diazo ketones, in the presence of the C=O group of various simple ketones and symrnetrical/unsymmetrical 1,2-diones were carried out. The reaction of alpha-diazo ketones with 1,2-naphthoquinone furnished interesting diastereomeric cycloadducts in which both the C=O groups acted as dipolarophilic sites. The similar reaction in the presence of several isatin derivatives afforded novel spiro dioxa-bridged indole derivatives as a mixture of diastereomers. The single crystal X-ray structure analysis manifestly revealed the mode of cycloaddition and the stereochemistry of two of the diastereomers. A diverse set of novel spiro epoxy-bridged tetrahydropyranone frameworks have been constructed in good yield via the tandem cyclization-cycloaddition of alpha-diazo ketones with the C=O group as heterodipolarophile in a regioselective manner. (C) 2003 Elsevier Ltd. All rights reserved.

Effect of ester chemical structure and peptide bond conformation in fragmentation pathways of differently metal cationized cyclodepsipeptides

Fragmentation behavior of two classes of cyclodepsipeptides, isariins and isaridins, obtained from the fungus Isaria, was investigated in the presence of different metal ions using multistage tandem mass spectrometry (MS(n)) with collision induced dissociation (CID) and validated by NMR spectroscopy. During MS(n) process, both protonated and metal-cationized isariins generated product ions belonging to the identical `b-ion' series, exhibiting initial backbone cleavage explicitly at the beta-ester bond. Fragmentation behavior for the protonated and metal-cationized acyclic methyl ester derivative of isariins was very similar. On the contrary, isaridins during fragmentation produced ions belonging to the `b' or/and the `y' ion series depending on the nature of interacting metal ions, due to initial backbone cleavages at the beta-ester linkage or/and at a specific amide linkage. Interestingly, independent of the nature of the interacting metal ions, the product ions formed from the acyclic methyl ester derivative of isaridins belonged only to the `y-type'. Complementary NMR data showed that, while all metal ions were located around the beta-ester group of isariins, the metal ion interacting sites varied across the backbone for isaridins. Combined MS and NMR data suggest that the different behavior in sequence specific charge-driven fragmentation of isariins and isaridins is predetermined because of the constituent beta-hydroxy acid residue in isariins and the cis peptide bond in isaridins.

(1S*,3S*,8S*,10S*)-10-Fluoro-15-oxatetracyclo[6.6.1.0(1,10).0( 3,8)]pentadeca-5,12-dien-3-ol

The title compound, C(14)H(17)FO(2), was obtained from anti-4a, 9a:8a,10a-diepoxy-1,4,4a,5,8,8a, 9,9a, 10,10a-decahydroanthracene via tandem hydrogen-fluoride-mediated epoxide ring-opening and transannular oxacyclization. With the two cyclohexene rings folded towards the oxygen bridge, the title tetracyclic fluoroalcohol molecule displays a conformation reminiscent of a pagoda. The crystal packing is effected via intermolecular O-H center dot center dot center dot O hydrogen bonds, which link the molecules into a zigzag chain along the b axis.

Structural, electrical, and optical properties of as-grown and heat treated ultra-thin SnS films

The composition, structural, electrical, and optical properties of as-grown and heat treated tin-mono-sulfide (SnS) ultra-thin films have been studied. The ultra-thin SnS films were prepared on glass substrates by thermal resistive evaporation technique. All the SnS films contained nanocrystallites and exhibited p-type conductivity with a low Hall-mobility, <50 cm(2)/Vs. All these films are highly tin rich in nature and exhibited orthorhombic crystal structure. As compared to other films, the SnS films annealed at 300 degrees C showed a low electrical resistivity of similar to 36 Omega cm with an optical band gap of similar to 1.98 eV. The observed electrical and optical properties of all the films are discussed based on their composition and structural parameters. These nanocrystalline ultra-thin SnS films could be expected as a buffer layer for the development of tandem solar cell devices due to their low-resistivity and high absorbability with an optimum band gap. (C) 2011 Elsevier B.V. All rights reserved.

Combined Electron Transfer Dissociation-Collision-Induced Dissociation Fragmentation in the Mass Spectrometric Distinction of Leucine, Isoleucine, and Hydroxyproline Residues in Peptide Natural Products

Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(center dot) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Delta m = 43 Da) or ethyl radicals (Delta m = 29 Da), through collisional activation of z(center dot) radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific e ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MSn) method has been successfully implemented in a liquid chromatography MSn platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.

A Unique Modification of the Eukaryotic Initiation Factor 5A Shows the Presence of the Complete Hypusine Pathway in Leishmania donovani

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N-(sic)-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of alpha-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A similar to 42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed similar to 40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite.