FTIR,FT-Raman spectra and quantum chemical studies of nebivolol

Fourier transform infrared and Raman spectra of nebivolol have been recorded. The structure, conformational stability, geometry optimisation, and vibrational wave numbers have been investigated. Satisfactory vibrational assignments were made for the stable conformer of the molecule using Restricted Hartree–Fock (RHF) and density functional theory (DFT) calculation (B3LYP) with the 6-31G(d,p) basis set. Comparison of the observed fundamental vibrational wave numbers of the molecule and calculated results by RHF and DFT methods indicates that B3LYP is superior for molecular vibrational problems. Comparison of the simulated spectra with the experimental spectra provides important information about the ability of the computational method to describe the vibrational modes. The RHF and DFT-based NMR calculation procedure was also done. It was used to assign the ¹³C NMR chemical shift of nebivolol.     

Computational and functional analysis of β-lactam resistance in Zymomonas mobilis

Zymomonas mobilis, a Gram-negative ethanologenic non-pathogenic bacterium, is reported to exhibit resistance to high concentrations of β-lactam antibiotics. In the present study, Z. mobilis was found to be resistant to I-IV generations of cephalosporins and carbapenems, i.e. narrow, broad and extended spectrum β-lactam antibiotics. We have analysed the genome of Z. mobilis (GenBank accession No.: NC 006526) harbouring multiple genes coding for β-lactamases (BLA), β-lactamase domain containing proteins (BDP) and penicillin binding proteins (PBP). The conserved domain database analysis of BDPs predicted them to be members of metallo β-lactamase superfamily. Further, class C specific multidomain AmpC (β-lactamase C) was found in the three β-lactamases. The β-lactam resistance determinants motifs, HXHXD, KXG, SXXK, SXN, and YXN are present in the BLAs, BDPs and PBPs of Z. mobilis. The predicted theoretical pI and aliphatic index values suggested their stability. One of the PBPs, PBP2, was predicted to share functional association with rod shape determining proteins (GenBank accession Nos. YP_162095 and YP_162091). Homology modelling of three dimensional structures of the β-lactam resistance determinants and further docking studies with penicillin and other β-lactam antibiotics indicated their substrate-specificity. Semi-quantitative PCR analysis indicated that the expression of all BLAs and one BDP are induced by penicillin. Disk diffusion assay, SDS-PAGE and zymogram analysis confirms the substrate specificity of the β-lactam resistance determinants. This study gives a broader picture of the β-lactam resistance determinants of a non-pathogenic ethanologenic Z. mobilis bacterium that could have implications in laboratories since it is routinely used in many research laboratories in the world for ethanol, fructooligosaccharides, levan production and has also been reported to be present in wine and beer as a spoilage organism.     

MICAL2 enhances branched actin network disassembly by oxidizing Arp3B-containing Arp2/3 complexes

The mechanisms regulating the disassembly of branched actin networks formed by the Arp2/3 complex still remain to be fully elucidated. In addition, the impact of Arp3 isoforms on the properties of Arp2/3 are also unexplored. We now demonstrate that Arp3 and Arp3B isocomplexes promote actin assembly equally efficiently but generate branched actin networks with different disassembly rates. Arp3B dissociates significantly faster than Arp3 from the network, and its depletion increases actin stability. This difference is due to the oxidation of Arp3B, but not Arp3, by the methionine monooxygenase MICAL2, which is recruited to the actin network by coronin 1C. Substitution of Arp3B Met293 by threonine, the corresponding residue in Arp3, increases actin network stability. Conversely, replacing Arp3 Thr293 with glutamine to mimic Met oxidation promotes disassembly. The ability of MICAL2 to enhance network disassembly also depends on cortactin. Our observations demonstrate that coronin 1C, cortactin, and MICAL2 act together to promote disassembly of branched actin networks by oxidizing Arp3B-containing Arp2/3 complexes.     

Molecular modeling and structural analysis of nAChR variants uncovers the mechanism of resistance to snake toxins

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.     

Controlled micelle conjugation via charged peptide amphiphiles

Abstract We report the first demonstration of nonionic detergent micelle conjugation and phase separation using purpose‐synthesized, peptide amphiphiles, C₁₀‐(Asp)₅ and C₁₀‐(Lys)₅. Clustering is achieved in two different ways. Micelles containing the negatively charged peptide amphiphile C₁₀‐(Asp)₅ are conjugated (a) via a water‐soluble, penta‐Lys mediator or (b) to micelles containing the C₁₀‐(Lys)₅ peptide amphiphile. Both routes lead to phase separation in the form of oil‐rich globules visible in the light microscope. The hydrophobic nature of these regions leads to spontaneous partitioning of hydrophobic dyes into globules that were found to be stable for weeks to months. Extension of the conjugation mechanism to micelles containing a recently discovered, light‐driven proton pump King Sejong 1‐2 (KS1‐2) demonstrates that a membrane protein may be concentrated using peptide amphiphiles while preserving its native conformation as determined by characteristic UV absorption. The potential utility of these peptide amphiphiles for biophysical and biomedical applications is discussed.     

Interdependence of backbone flexibility,residue conservation,and enzyme function: a case study on beta1,4-galactosyltransferase-I

Beta1,4-galactosyltransferase-I (beta4Gal-T1) catalyzes the transfer of a galactose from UDP-galactose to N-acetylglucosamine. A recent crystal structure determination of the substrate-bound enzyme reveals a large conformational change, which creates binding sites for the oligosaccharide and alpha-lactalbumin, when compared to the ligand-free structure. The conformational changes take place in a 21-residue-long loop (I345-H365) and in a smaller loop containing a tryptophan residue (W314) flanked by glycines (Y311-G316; Trp loop). A series of molecular dynamics simulations carried out with an implicit solvent model and with explicit water successfully identify flexibility in the two loops and in another interacting loop. These observations are confirmed by limited proteolysis experiments that reveal an intrinsic flexibility of the long loop. The multiple simulation runs starting with the substrate-free structure show that the long loop moves toward its conformation in the ligand-bound structure; however, it gets stabilized in an intermediate position. The Trp loop moves in the opposite direction to that of the long loop, making contacts with residues in the long loop. Remarkably, when the Trp loop is restrained in its starting conformation, no large conformational change takes place in the long loop, indicating residue communication of flexibility. Sequence and structural analysis of the beta4Gal-T1 family with 37 known sequences reveals that in contrast to the unconserved long loop, which undergoes a much larger conformational change, the Trp loop including the glycines is highly conserved. These observations lead us to propose a new functional mechanism that may be conserved by evolution to perform a variety of functions.     

Requirement for direct association of ammonia-excreting Anabaena variabilis mutant (SA-1) with roots for maximal growth and yield of wheat

Direct association between wheat roots and an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis, strain SA-1, was required for maximal enhancement of growth of wheat plants in nitrogen (N)-free, hydroponic medium. Over 85% of the cyanobacterial mutant SA-1 inoculated to the roots were adsorbed under non-saturating conditions. The adsorption process of SA-1 to wheat roots was biphasic: an initial rapid adsorption was followed by a slow phase with about 10% of the initial adsorption rate. The maximal adsorption rate of filaments observed was 1.6 mg dry wt. SA-1 adsorbed·plant^–1·h^–1. Bypassing CO₂ fixation and sugar formation, the ¹⁴C label from [¹⁴C]sucrose was directly applied to leaf blades to study sugar translocation. The ¹⁴C label from this treatment appeared in the wheat culture medium within an hour. Nitrate-grown plants excreted about 30% of the ¹⁴C label into the medium, compared to only 10% excreted by wheat/Anabaena co-cultures. SA-1 assimilated 27% of all ¹⁴C translocated from [U-¹⁴C]sucrose applied to wheat leaves, and ¹⁴C label from this treatment was recovered from strain SA-1 after 30 min. Roots and cyanobacteria accounted for 51% of all radioactive label recovered in the plants co-cultured with SA-1 vs 20% for nitrate-grown plants. We studied the activity of -fructosidase (invertase) in wheat of variety Yecora rojo. Roots from nitrate-grown wheat plants produced high levels of invertase activity, which converted over 85% of 3 mm sucrose into glucose and fructose in 24 h. The rate of sucrose disappearance in the medium of co-cultures using A. variabilis SA-1, was 70% of that of nitrate-grown plants, but the levels of glucose and fructose in these cultures were always very low during sucrose conversion, suggesting hexose assimilation. To study the role of diffusible metabolites, a dialysis membrane was employed to separate the ammonia-excreting SA-1 from the wheat roots. Containing SA-1 in a dialysis bag away from direct root contact, severely limited leaf growth to less than one-third of the growth rate of nitrate control cultures. Ammonia produced by mutant SA-1 in dialysis bag cultures was excreted into the medium at 0.4 mm vs 1.2 mm in free-living cultures, but ammonia was not detectable in co-cultures with or without the dialysis bag containing the mutant. The nitrogenase activity derepressed in the mutant and responsible for ammonia excretion was always higher in the association co-cultures than in either free cells or in dialysis-bag cultures. The nitrogenase activity of strain SA-1 was highest (200 mol ethylene formed·mg^–1 Chl·h^–1) when the cyanobacterium was associated with the root tips. Dialysis membrane separation of plant and cyanobacterium severely inhibited growth of wheat during a complete growth cycle of 2 months. Total biomass and grain yield were very similar for control cultures without inorganic N or SA-1, and for diffusion cultures containing SA-1, kept in a dialysis bag around the roots. Total biomass of the association co-culture attained 75% of the biomass of the nitrate-grown control. It is proposed that wheat roots supplied fructose derived from sucrose for growth and nitrogen fixation of SA-1 in the light, and that ammonia excreted by SA-1 was utilized by the wheat plant for its own growth. Correspondence to: H. Spiller     

Simultaneous oxygen production and nitrogenase activity of an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis in a co-culture with wheat

Summary A mutant strain of Anabaena variabilis, strain SA-1 that supported growth of wheat plants in a hydroponic co-culture in nitrogen (N) free medium also produced enough oxygen (O₂) to support root respiration. The steady-state concentration of net O₂ in the co-culture was dependent on incident light intensity. At an incident photosynthetic photoflux (PPF) of 1000 mol photons·m^–2·s^–1, net O₂ evolution by the co-culture in the root zone reached a maximum value of about 220 mol O₂ evolved·h^–1·mg chlorophyll (Chl)^–1. The O₂ concentration in the rhizosphere of the co-culture stayed above the ambient air level. O₂ uptake in the dark by strain SA-1-supplemented wheat roots washed free of cyanobacterium was higher than the root respiration of nitrate-grown plants. Nitrate-grown plants required aeration for maximum growth while the wheat-cyanobacterial co-culture can be cultured without aeration. These results show that O₂ produced by strain SA-1 can be used to supply the O₂ needs for root respiration of wheat. Respiration reduced net O₂ evolution by the mutant SA-1, decreasing the partial pressure of O₂ at the sites of cyanobacterial attachment to the roots. This led to an increase in the specific activity of nitrogenase of the co-culture at the high light intensities used to support wheat growth. This activity of about 30 mol ethylene produced·mg Chl^–1·h^–1 was three-fold higher than the activities obtained with the free-living strain SA-1 assayed at the same light intensity. In the co-culture, ammonia produced by the mutant strain SA-1 was not detectable. The NH _inf4 ^sup+ produced by strain SA-1 was used by the wheat plants and, under these conditions, the total N content of the plants reached as high as 85% of the total N content of nitrate-grown plants. In the co-culture system the metabolism of wheat and the cyanobacterium complemented each other, leading to higher plant growth in N-free medium. Offprint requests to: M. Gunasekaran