Endonuclease Active Site Plasticity Allows DNA Cleavage with Diverse Alkaline Earth and Transition Metal Ions

A majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion.

Present status of Gottigere Tank : Indicator of Decision maker’s apathy

Gottigere lake with a water spread area of about 14.98 ha is located in the Bellandur Lake catchment of the South Pennar River basin. In recent years, this lake catchment has been subjected to environmental stress mainly due to the rampant unplanned developmental activities in the catchment. The functional ability of the ecosystem is impaired due to structural changes in the ecosystem. This is evident from poor water quality, breeding of disease vectors, contamination of groundwater in the catchment, frequent flooding in the catchment due to topography alteration, decline in groundwater table, erosion in lake bed, etc. The development plans of the region (current as well as the proposed) ignore the integrated planning approaches considering all components of the ecosystem. Serious threats to the sustainability of the region due to lack of holistic approaches in aquatic resources management are land use changes (removal of vegetation cover, etc.), point and non-point sources of pollution impairing water quality, dumping of solid waste (building waste, etc.). Conservation of lake ecosystem is possible only when the physical and chemical integrity of its catchment is maintained. Alteration in the catchment either due to land use changes (leading to paved surface area from vegetation cover), alteration in topography, construction of roads in the immediate vicinity are detrimental to water yield in the catchment and hence, the sustenance of the lake. Open spaces in the form of lakes and parks aid as kidney and lung in an urban ecosystem, which maintain the health of the people residing in the locality. Identification of core buffer zones and conservation of buffer zones (500 to 1000 m from shore) is to be taken up on priority for conservation and sustainable management of Bangalore lakes. Bangalore is located over a ridge delineating four watersheds, viz. Hebbal, Koramangala, Challaghatta and Vrishabhavathi. Lakes and tanks are an integral part of natural drainage and help in retaining water during rainfall, which otherwise get drained off as flash floods. Each lake harvests rainwater from its catchment and surplus flows downstream spilling into the next lake in the chain. The topography of Bangalore has uniquely supported the creation of a large number of lakes. These lakes form chains, being a series of impoundments across streams. This emphasises the interconnectivity among Bangalore lakes, which has to be retained to prevent Bangalore from flooding or from water scarcity. The main source of replenishment of groundwater is the rainfall. The slope of the terrain allows most of the rainwater to flow as run-off. With the steep gradients available in the major valleys of Bangalore, the rainwater will flow out of the city within four to five hours. Only a small fraction of the rainwater infiltrates into the soil. The infiltration of water into the subsoil has declined with more and more buildings and paved road being constructed in the city. Thus the natural drainage of Bangalore is governed by flows from the central ridge to all lower contours and is connected with various tanks and ponds. There are no major rivers flowing in Bangalore and there is an urgent need to sustain these vital ecosystems through proper conservation and management measures. The proposed peripheral ring road connecting Hosur Road (NH 7) and Mysore Road (SH 17) at Gottigere lake falls within the buffer zone of the lake. This would alter the catchment integrity and hence water yield affecting flora, fauna and local people, and ultimately lead to the disappearance of Gottigere lake. Developmental activities in lake catchments, which has altered lake’s ecological integrity is in violation of the Indian Fisheries Act – 1857, the Indian Forest Act – 1927, Wildlife (Protection) Act – 1972, Water (Prevention and Control of Pollution) Act – 1974, Water (Prevention and Control of Pollution) Act – 1977, Forest (Conservation Act) – 1980, Environmental (Protection) Act – 1986, Wildlife (Protection) Amendment Act – 1991 and National Conservation Strategy and Policy Statement on Environment and Development – 1992. Considering 65% decline of waterbodies in Bangalore (during last three decades), decision makers should immediately take preventive measures to ensure that lake ecosystems are not affected. This report discusses the impacts due to the proposed infrastructure developmental activities in the vicinity of Gottigere tank.

Robust Fixed Order Lateral H2 Controller for Micro Air Vehicle

This paper presents a robust fixed order H2controller design using strengthened discrete optimal projection equations, which approximate the first order necessary optimality condition. The novelty of this work is the application of the robust H2controller to a micro aerial vehicle named Sarika2 developed in house. The controller is designed in discrete domain for the lateral dynamics of Sarika2 in the presence of low frequency atmospheric turbulence (gust) and high frequency sensor noise. The design specification includes simultaneous stabilization, disturbance rejection and noise attenuation over the entire flight envelope of the vehicle. The resulting controller performance is comprehensively analyzed by means of simulation

Relation between Local Structure and Glass Forming Ability of Liquid Alloys

An attempt has been made to describe the glass forming ability (GFA) of liquid alloys, using the concepts of the short range order (SRO) and middle range order (MRO) characterizing the liquid structure.A new approach to obtain good GFA of liquid alloys is based on the following four main factors: (1) formation of new SRO and competitive correlation with two or more kinds of SROs for crystallization, (2) stabilization of dense random packing by interaction between different types of SRO, (3) formation of stable cluster (SC) or middle range order (MRO) by harmonious coupling of SROs, and (4) difference between SRO characterizing the liquid structure and the near-neighbor environment in the corresponding equilibrium crystalline phases. The atomic volume mismatch estimated from the cube of the atomic radius was found to be a close relation with the minimum solute concentration for glass formation. This empirical guideline enables us to provide the optimum solute concentration for good GFA in some ternary alloys. Model structures, denoted by Bernal type and the Chemical Order type, were again tested in the novel description for the glass structure as a function of solute concentration. We illustrated the related energetics of the completion between crystal embryo and different types of SRO. Recent systematic measurements also provide that thermal diffusivity of alloys in the liquid state may be a good indicator of their GFA.

Structure of the Pyridine-Chloranil Complex in Solution: A Surprise from Depolarized Hyper-Rayleigh Scattering Measurements

In this article, we report the structure of a 1:1 charge transfer complex between pyridine (PYR) and chloranil (CHL) in solution (CHCl(3)) from the measurement of hyperpolarizability (beta(HRS)) and linear and circular depolarization ratios, D and D', respectively, by the hyper-Rayleigh scattering technique and state-of-the-art quantum chemical calculations. Using linearly (electric field vector along X) and circularly polarized incident light, respectively, we have measured two macroscopic depolarization ratios D = I(X,X)(2 omega)/I(X,Z)(2 omega) and D' = I(X,C)(2 omega)/I(Z,C)(2 omega) in the laboratory fixed XYZ frame by detecting the second harmonic (SH) scattered light in a polarization resolved fashion. The stabilization energy and the optical gap calculated through the MP2/cc-pVDZ method using Gaussian09 were not significantly different to distinguish between the cofacial and T-shape structures. Only when the experimentally obtained beta(HRS) and the depolarization ratios, D and D', were matched with the theoretically computed values from single and double configuration interaction (SDCI) calculations performed using the ZINDO-SCRF technique, we concluded that the room temperature equilibrium structure of the complex is cofacial. This is in sharp contrast to an earlier theoretical prediction of the T-shape structure of the complex.

Interaction of G-Quadruplexes with Nonintercalating Duplex-DNA Minor Groove Binding Ligands

The enzyme telomerase synthesizes the G-rich DNA strands of the telomere and its activity is often associated with cancer. The telomerase may be therefore responsible for the ability of a cancer cell-to escape apoptosis. The G-rich DNA sequences often adopt tetra-stranded structure, known as the G-quadruplex DNA (G4-DNA). The stabilization of the telomeric DNA into the G4-DNA structures by small molecules has been the focus of many researchers for the design and development of new anticancer agents. The compounds which stabilize the G-quadruplex in the telomere inhibit the telomerase activity. Besides telomeres, the G4-DNA forming sequences are present in the genomic regions of biological significance including the transcriptional regulatory and promoter regions of several oncogenes. Inducing a G-quadruplex structure within the G-rich promoter sequences is a potential way of achieving selective gene regulation. Several G-quadruplex stabilizing ligands are known. Minor groove binding ligands (MGBLs) interact with the double-helical DNA through the minor grooves sequence-specifically and interfere with several DNA associated processes. These MGBLs when suitably modified switch their preference sometimes from the duplex DNA to G4-DNA and stabilize the G4-DNA as well. Herein, we focus on the recent advances in understanding the G-quadruplex structures, particularly made by the human telomeric ends, and review the results of various investigations of the interaction of designed organic ligands with the G-quadruplex DNA while highlighting the importance of MGBL-G-quadruplex interactions.

NMR Structure Implications of Enhanced Efficacy of Obestatin Fragment Analogs

Obestatin is a more recently discovered hormone that is encoded by the ghrelin gene and produced in the stomach and gut. We report NMR analysis on synthetic Obestatin (OB23), a 23 residue peptide, along with three overlapping fragments of the same in methanol solvent as a first step towards structure activity relationship. Selective substitutions on the promising N-terminal and middle fragments of obestatin have been carried out in order to improve the efficacy and potency. In the N-terminal fragment two peptides were obtained by the replacement of Gly (8) with a-aminoisobutyric acid (Aib, U) and Phe (F5) with Cyclohexylalanine (Cha). In case of the middle fragment both Gly (3) and Gly (8) were replaced with Aib residues. The rationale being, these unusual amino acids could provide protection from immediate degradation and aid structure stabilization. Our previous studies showed that the N-terminal and the middle fragment were unstructured and hence this substitution would directly evaluate the effect of structure on the activity of these fragment analogs. Detailed NMR analysis clearly demonstrates formation of helical secondary structure in all the peptide analogues and provides justification for relative activities reported by our group previously (Nagaraj et al. 2009).

Roles of residues in the interface of transient protein-protein complexes before complexation

Transient protein-protein interactions play crucial roles in all facets of cellular physiology. Here, using an analysis on known 3-D structures of transient protein-protein complexes, their corresponding uncomplexed forms and energy calculations we seek to understand the roles of protein-protein interfacial residues in the unbound forms. We show that there are conformationally near invariant and evolutionarily conserved interfacial residues which are rigid and they account for similar to 65% of the core interface. Interestingly, some of these residues contribute significantly to the stabilization of the interface structure in the uncomplexed form. Such residues have strong energetic basis to perform dual roles of stabilizing the structure of the uncomplexed form as well as the complex once formed while they maintain their rigid nature throughout. This feature is evolutionarily well conserved at both the structural and sequence levels. We believe this analysis has general bearing in the prediction of interfaces and understanding molecular recognition.

Dimeric 1,3-Phenylene-bis(piperazinyl benzimidazole)s: Synthesis and Structure-Activity Investigations on their Binding with Human Telomeric G-Quadruplex DNA and Telomerase Inhibition Properties

Ligand-induced stabilization of G-quadruplex structures formed by the human telomeric DNA is an active area of research. The compounds which stabilize the G-quadruplexes often lead to telomerase inhibition. Herein we present the results of interaction of new monomeric and dimeric ligands having 1,3-phenylene-bis(piperazinyl benzimidazole) unit with G-quadruplex DNA (G4DNA) formed by human telomeric repeat d(G(3)T(2)A)(3)G(3)]. These ligands efficiently stabilize the preformed G4DNA in the presence of 100 mM monovalent alkali metal ions. Also, the G4DNA formed in the presence of low concentrations of ligands in 100 mM K+ adopts a highly stable parallel-stranded conformation. The G-quadruplexes formed in the presence of the dimeric compound are more stable than that induced by the corresponding monomeric counterpart. The dimeric ligands having oligo-oxyethylene spacers provide much higher stability to the preformed G4DNA and also exert significantly higher telomerase inhibition activity. Computational aspects have also been discussed.

Electromechanical dynamics and optimization of pectoral fin-based ionic polymer-metal composite underwater propulsor

Ionic polymer-metal composites are soft artificial muscle-like bending actuators, which can work efficiently in wet environments such as water. Therefore, there is significant motivation for research on the development and design analysis of ionic polymer-metal composite based biomimetic underwater propulsion systems. Among aquatic animals, fishes are efficient swimmers with advantages such as high maneuverability, high cruising speed, noiseless propulsion, and efficient stabilization. Fish swimming mechanisms provide biomimetic inspiration for underwater propulsor design. Fish locomotion can be broadly classified into body and/or caudal fin propulsion and median and/or paired pectoral fin propulsion. In this article, the paired pectoral fin-based oscillatory propulsion using ionic polymer-metal composite for aquatic propulsor applications is studied. Beam theory and the concept of hydrodynamic function are used to describe the interaction between the beam and water. Furthermore, a quasi-steady blade element model that accounts for unsteady phenomena such as added mass effects, dynamic stall, and the cumulative Wagner effect is used to obtain hydrodynamic performance of the ionic polymer-metal composite propulsor. Dynamic characteristics of ionic polymer-metal composite fin are analyzed using numerical simulations. It is shown that the use of optimization methods can lead to significant improvement in performance of the ionic polymer-metal composite fin.