Fluorescence quenching of biologically active carboxamide by aniline and carbon tetrachloride in different solvents using Stern-Volmer plots

Fluorescence quenching of biologically active carboxamide namely (E)-2-(4-chlorobenzylideneamino)-N-(2-chlorophenyl)-4,5,6,7-tetrahydrobe nzo[b]thiophene-3-carboxamide [ECNCTTC] by aniline and carbon tetrachloride (CCl(4)) quenchers in different solvents using steady state method and time resolved method using only one solvent has been carried out at room temperature to understand the role of quenching mechanisms. The Stern-Volmer plot has been found to be linear for all the solvents studied. The probability of quenching per encounter p (p') was determined in all the solvents and was found to be less than unity. Further, from the studies of rate parameters and life time measurements in n-heptane and cyclohexane with aniline and carbon tetrachloride as quenchers have been shown that, the phenomenon of quenching is generally governed by the well-known Stern-Volmer (S-V) plot. The activation energy E(a) (or E(a)') of quenching was determined using the literature values of activation energy of diffusion E(d) and the experimentally determined values of p (or p'). It has been found that, the activation energy E(a) (E(a)') is greater than the activation energy for diffusion E(d) in all solvents. Hence, from the magnitudes of E(a) (or E(a)') as well as p (or p') infer that, the quenching mechanism is not solely due to the material diffusion, but there is also contribution from the activation energy. (C) 2011 Elsevier B.V. All rights reserved.

Solvent effect on the fluorescence quenching of biologically active carboxamide by aniline and carbon tetrachloride in different solvents using S-V plots

The fluorescence quenching studies of carboxamide namely (E)-N-(3-Chlorophenyl)-2-(3,4,5-trimethoxybenzylideneamino)-4,5,6,7 tetrahydrobenzob]thiophene-3-carboxamide ENCTTTC] by aniline and carbon tetrachloride in six different solvents namely toluene, cyclohexane, n-hexane, n-heptane, n-decane and n-pentane have been carried out at room temperature with a view to understand the quenching mechanisms. The Stern-Volmer (S-V) plots have been found to be nonlinear with a positive deviation for all the solvents studied. In order to interpret these results we have invoked the ground state complex formation and sphere of action static quenching models. Using these models various quenching rate parameters have been determined. The magnitudes of these parameters suggest that sphere of action static quenching model agrees well with the experimental results. Hence the positive deviation is attributed to the static and dynamic quenching. Further, with the use of Finite Sink approximation model, it was possible to check these bimolecular reactions as diffusion-limited and to estimate independently distance parameter R' and mutual diffusion coefficient D. Finally an effort has been made to correlate the values of R' and D with the values of the encounter distance R and the mutual coefficient D determined using the Edward's empirical relation and Stokes Einstein relation. (C) 2011 Elsevier B.V. All rights reserved.

Co-liposomes of redox-active alkyl-ferrocene modified low MW branched PEI and DOPE for efficacious gene delivery in serum

Herein, we present six new lipopolymers based on low molecular weight, branched polyethylenimine (BPEI 800 Da) which are hydrophobically modified using ferrocene terminated alkyl tails of variable lengths. The effects of degree of grafting, spacer length and the redox state of ferrocene in the lipopolymers on the self assembly properties were investigated in detail by TEM, AFM, DLS and zeta potential measurements. The assemblies displayed an oxidation induced increase in the size of the aggregates. The co-liposomes comprising the lipopolymer and a helper lipid, 1,2-dioleoyl phosphatidyl ethanolamine (DOPE), showed excellent gene (pDNA) delivery capability in a serum containing environment in two cancer cell lines (HeLa and U251 cells). Optimized formulations showed remarkably higher transfection activity than BPEI (25 kDa) and were also significantly better than a commercial transfection reagent, Lipofectamine 2000 as evidenced from both the luciferase activity and GFP expression analysis. Oxidation of ferrocene in the lipopolymers led to drastically reduced levels of gene transfection which was substantiated by reduced cellular internalization of fluorescently labelled pDNA as detected using confocal microscopy and flow cytometry. Moreover, the transfection inactive oxidized lipopolyplexes could be turned transfection active by exposure to ascorbic acid (AA) in cell culture medium during transfection. Endocytosis inhibition experiments showed that gene expression mediated by reduced formulations involved both clathrin and caveolae mediated pathways while the oxidized formulations were routed via the caveolae. Cytotoxicity assays revealed no obvious toxicity for the lipopolyplexes in the range of optimized transfection levels in both the cell lines studied. Overall, we have exploited the redox activity of ferrocene in branched PEI-based efficient polymeric gene carriers whose differential transfection activities could be harnessed for spatial or temporal cellular transfections.

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalentlv associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hGG alpha and hCG beta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Go-expression of both subunits in the same cell resulted in secretion of heterodimeric hGG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hGG. The le level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermenter. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hGG and is suitable for structure-function studies of the hormone.

Structural Basis of Drug Resistance by Genetic Variants of HIV Type 1 Clade C Protease from India

Using computer modeling of three-dimensional structures and structural information available on the crystal structures of HIV-1 protease, we investigated the structural effects of mutations, in treatment-naive and treatment-exposed individuals from India and postulated mechanisms of resistance in clade C variants. A large number of models (14) have been generated by computational mutation of the available crystal structures of drug bound proteases. Localized energy minimization was carried out in and around the sites of mutation in order to optimize the geometry of interactions present. Most of the mutations result in structural differences at the flap that favors the semiopen state of the enzyme. Some of the mutations were also found to confer resistance by affecting the geometry of the active site. The E35D mutation affects the flap structure in clade B strains and E35N and E35K mutation, seen in our modeled strains, have a more profound effect. Common polymorphisms at positions 36 and 63 in clade C also affected flap structure. Apart from a few other residues Gln-58, Asn-83, Asn-88, and Gln-92 and their interactions are important for the transition from the closed to the open state. Development of protease inhibitors by structure-based design requires investigation of mechanisms operative for clade C to improve the efficacy of therapy.

A theoretical model for the magnetic helicity of solar active regions

Active regions on the solar surface are known to possess magnetic helicity, which is predominantly negative in the northern hemisphere and positive in the southern hemisphere. Choudhuri et al. [Choudhuri, A.R. On the connection between mean field dynamo theory and flux tubes. Solar Phys. 215, 31–55, 2003] proposed that the magnetic helicity arises due to the wrapping up of the poloidal field of the convection zone around rising flux tubes which form active regions. Choudhuri [Choudhuri, A.R., Chatterjee, P., Nandy, D. Helicity of solar active regions from a dynamo model. ApJ 615, L57–L60, 2004] used this idea to calculate magnetic helicity from their solar dynamo model. Apart from getting broad agreements with observational data, they also predict that the hemispheric helicity rule may be violated at the beginning of a solar cycle. Chatterjee et al. [Chatterjee, P., Choudhuri, A.R., Petrovay, K. Development of twist in an emerging magnetic flux tube by poloidal field accretion. A&A 449, 781–789, 2006] study the penetration of the wrapped poloidal field into the rising flux tube due to turbulent diffusion using a simple 1-d model. They find that the extent of penetration of the wrapped field will depend on how weak the magnetic field inside the rising flux tube becomes before its emergence. They conclude that more detailed observational data will throw light on the physical conditions of flux tubes just before their emergence to the photosphere.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Structures and molecular-dynamics studies of three active-site mutants of bovine pancreatic phospholipase A(2)

Phospholipase A(2) hydrolyzes phospholipids at the sn-2 position to cleave the fatty-acid ester bond of L-glycerophospholipids. The catalytic dyad (Asp99 and His48) along with a nucleophilic water molecule is responsible for enzyme hydrolysis. Furthermore, the residue Asp49 in the calcium-binding loop is essential for controlling the binding of the calcium ion and the catalytic action of phospholipase A2. To elucidate the structural role of His48 and Asp49, the crystal structures of three active-site single mutants H48N, D49N and D49K have been determined at 1.9 angstrom resolution. Although the catalytically important calcium ion is present in the H48N mutant, the crystal structure shows that proton transfer is not possible from the catalytic water to the mutated residue. In the case of the Asp49 mutants, no calcium ion was found in the active site. However, the tertiary structures of the three active-site mutants are similar to that of the trigonal recombinant enzyme. Molecular-dynamics simulation studies provide a good explanation for the crystallographic results.

Specificity for the Exchange of Phospholipids Through Polymyxin B Mediated Intermembrane Molecular Contacts

Structural specificity for the direct vesicle−vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O. G., & Jain, M. K. (1996) Biochemistry 35, 299−308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle−vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange in covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that stable PxB-mediated vesicle−vesicle contacts may be structurally and functionally distinct from “stalks”, the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis and trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundations of interfacial equilibria and catalysis.

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.

Cloning,overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from scherichia coli

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. (C) 2010 Elsevier Inc. All rights reserved.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn2+, at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent crosslinking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

High-Resolution Magic Angle Spinning Nmr-Studies Of P-31 And B-11 In Fast Ion Conducting Agi-Ag2o-P2o5 And Agi-Ag2o-B2o3 Glasses

Silver iodide-based fast ion conducting glasses containing silver phosphate and silver borate have been studied. An attempt is made to identify the interaction between anions by studying the chemical shifts of31P and11B atoms in high resolution (HR) magic angle spinning (MAS) NMR spectra. Variation in the chemical shifts of31P or11B has been observed which is attributed to the change in the partial charge on the31P or11B. This is indicative of the change in the electronegativity of the anion matrix as a whole. This in turn is interpreted as due to significant interaction among anions. The significance of such interaction to the concept of structural unpinning of silver ions in fast ion conducting glasses is discussed.

Developmental regulation of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) gene expression in rat liver

The expression of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes has been studied as a function of development in rat liver. The levels of cytochrome P-450 (b+e) mRNAs and their transcription rates are too low for detection in the 19-day old fetal liver before or after phenobarbitone treatment. However, glutathione transferase (Ya+Yc) mRNAs can be detected in the fetal liver as well as their induction after phenobarbitone treatment can be demonstrated. These mRNAs contents as well as their inducibility with phenobarbitone are lower in maternal liver than that of adult nonpregnant female rat liver. Steroid hormone administration to immature rats blocks substantially the phenobarbitone mediated induction of the two mRNA families as well as their transcription. It is suggested that steroid hormones constitute one of the factors responsible for the repression of the cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes in fetal liver.