Crystal and molecular structure of putrescine DL- glutamic acid complex

The polyamines spermine, spermidine, putrescine, cadaverine, etc. have been implicated in a variety of cellular functions. However, details of their mode of interaction with other ubiquitous biomolecules is not known. We have solved a few structures of polyamine-amino acid complexes to understand the nature and mode of their interactions. Here we report the structure of a complex of putrescine with DL-glutamic acid. Comparison of the structure with the structure of putrescine-L-glutamic acid complex reveals the high degree of similarity in the mode of interaction in the two complexes. Despite the presence of a centre of symmetry in the present case, the arrangement of molecules is strikingly similar to the L-glutamic acid complex.

The crystal and molecular structure of putrescine - di-glutamic acid complexes

The polyamines spermine, spermidine, putrescine, cadaverine, etc. have been implicated in a variety of cellular functions. However, details of their mode of interaction with other ubiquitous biomolecules is not known. We have solved a few structures of polyamine-amino acid complexes to understand the nature and mode of their interactions. Here we report the structure of a complex of putrescine with DL-glutamic acid. Comparison of the structure with the structure of putrescine-L-glutamic acid complex reveals the high degree of similarity in the mode of interaction in the two complexes. Despite the presence of a centre of symmetry in the present case, the arrangement of molecules is strikingly similar to the L-glutamic acid complex.

Structure-Activity Relationship of Photocytotoxic Iron(III) Complexes of Modified Dipyridophenazine Ligands

Iron(III) complexes FeL(B)] (1-5) of a tetradentate trianionic phenolate-based ligand (L) and modified dipyridophenazine bases (B), namely, dipyrido-6,7,8,9-tetrahydrophenazine (dpqC in 1), dipyrido3,2-a:2',3'-c]phenazine-2-carboxylic acid (dppzc in 2), dipyrido3,2-a:2',3'-c]phenazine-11-sulfonic acid (dppzs in 3), 7-aminodipyrido3,2-a:2',3'-c]phenazine (dppza in 4) and benzoi]dipyridro3,2-a:2',3'-c]phenazine (dppn in 5), have been synthesized, and their photocytotoxic properties studied along with their dipyridophenazine analogue (6). The complexes have a five. electron paramagnetic iron(III) center, and the Fe(III)/Fe(II) redox couple appears at about 0.69 V versus SCE in DMF-0.1 M TBAP. The physicochemical data also suggest that the complexes possess similar structural features as that of its parent complex FeL(dppz)] with FeO3N3 coordination in a distorted octahedral geometry. The DNA-complex and protein-complex interaction studies have revealed that the complexes interact favorably with the biomolecules, the degree of which depends on the nature of the substituents present on the dipyridophenazine ring. Photocleavage Of pUC19 DNA by the complexes has been studied using visible light of 476, 530, and 647 nm wavelengths. Mechanistic investigations with inhibitors show formation of HO center dot radicals via a photoredox pathway. Photocytotoxicity study of the complexes in HeLa cells has shown that the dppn complex (5) is highly active in causing cell death in visible light with sub micromolar IC50 value. The effect of substitutions and the planarity of the phenazine moiety on the cellular uptake are quantified by determining the total Cellular iron content using the inductively coupled plasma-optical emission spectrometry (ICP-OES) technique. The cellular uptake increases marginally with an increase in the hydrophobicity of the dipyridophenazine ligands whereas complex 3 with dppzs shows very high uptake. Insights into the cell death mechanism by the dppn complex 5, obtained through DAFT nuclear staining in HeLa cells, reveal a rapid programmed cell death mechanism following photoactivation of complex 5 with visible light. The effect of substituent on the DNA photocleavage activity of the complexes has been rationalized from the theoretical studies.

Baculovirus mediated high-level expression of luciferase in silkworm cells and larvae

Bombyx mori nuclear polyhedrosis virus (BmNPV)-based baculovirus expression system exploits silkworm larvae as an economical alternative to large-scale cell cultures for production of biomolecules. To generate recombinant BmNPV at high efficiency, we have achieved high efficiency transfection of B. mori cells, BmN, through lipofection. Optimal conditions for lipofection were standardized by quantification of the transient expression level of firefly luciferase (luc) reporter gene under control of an immediate early gene promoter of BmNPV Lipofection was 50-fold and 100-fold more efficient than the calcium phosphate method for transfecting BmN and Sf9 cells, respectively. Lipofection enabled us to generate a recombinant BmNPV (vBmluc), harboring luc under control of the strong polyhedrin promoter On infection with vBmluc, luciferase was expressed at very high levels, 170 mu g/10(6) BmN cells or 13 mg/larva. Expression of luciferase in vBmluc-infected larvae was visualized by luminescence emission instantaneously following luciferin injection generating ''glowing silkworms''.

Biopolymers in nanopores: challenges and opportunities

We review the current status of various aspects of biopolymer translocation through nanopores and the challenges and opportunities it offers. Much of the interest generated by nanopores arises from their potential application to third-generation cheap and fast genome sequencing. Although the ultimate goal of single-nucleotide identification has not yet been reached, great advances have been made both from a fundamental and an applied point of view, particularly in controlling the translocation time, fabricating various kinds of synthetic pores or genetically engineering protein nanopores with tailored properties, and in devising methods (used separately or in combination) aimed at discriminating nucleotides based either on ionic or transverse electron currents, optical readout signatures, or on the capabilities of the cellular machinery. Recently, exciting new applications have emerged, for the detection of specific proteins and toxins (stochastic biosensors), and for the study of protein folding pathways and binding constants of protein-protein and protein-DNA complexes. The combined use of nanopores and advanced micromanipulation techniques involving optical/magnetic tweezers with high spatial resolution offers unique opportunities for improving the basic understanding of the physical behavior of biomolecules in confined geometries, with implications for the control of crucial biological processes such as protein import and protein denaturation. We highlight the key works in these areas along with future prospects. Finally, we review theoretical and simulation studies aimed at improving fundamental understanding of the complex microscopic mechanisms involved in the translocation process. Such understanding is a pre-requisite to fruitful application of nanopore technology in high-throughput devices for molecular biomedical diagnostics.

Bio-Composite Membrane Electrolytes for Direct Methanol Fuel Cells

A new class of bio-composite polymer electrolyte membranes comprising chitosan (CS) and certain biomolecules in particular, plant hormones such as 3-indole acetic acid (IAA), 4-chlorophenoxy acetic acid (CAA) and 1-naphthalene acetic acid (NAA) are explored to realize proton-conducting bio-composite membranes for application in direct methanol fuel cells (DMFCs). The sorption capability, proton conductivity and ion-exchange capacity of the membranes are characterized in conjunction with their thermal and mechanical behaviour. A novel approach to measure the permeability of the membranes to both water and methanol is also reported, employing NMR imaging and volume localized NMR spectroscopy, using a two compartment permeability cell. A DMFC using CS-IAA composite membrane, operating with 2M aqueous methanol and air at 70 degrees C delivers a peak power density of 25 mW/cm(2) at a load current density of 150 mA/cm(2). The study opens up the use of bio-compatible membranes in polymer-electrolyte-membrane fuel cells. (C) 2011 The Electrochemical Society. [DOI: 10.1149/2.030111jes] All rights reserved.

Structure and dynamics of protein excited states with millisecond lifetimes

An in-depth understanding of biological processes often requires detailed atomic resolution structures of the molecules involved. However in solution where most of these processes occur the conformation of biomolecules like RNA, DNA and proteins is not static but fluctuates. Routinely used structural techniques like X-ray crystallography, NMR spectroscopy and cryo-electron microscopy have almost always been used to determine the structure of the dominant conformation or obtain an average structure of the biomolecule in solution with very little detailed information regarding the dynamics of these molecules in solution. Over the last few years, NMR based methods have been developed to study the dynamics of these biomolecules in solution in a site-specific manner with the aim of generating structures of the different conformations that these molecules can adopt in solution. One powerful technique is the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment, which can be used to detect and characterize protein excited states that are populated for as less as 0.5% of the time with ∼0.5–10 millisecond lifetimes. Due to recent advances in NMR pulse sequences and labeling methodology, it is now possible to determine the structures of these transiently populated excited states with millisecond lifetimes by obtaining accurate chemical shifts, residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) of these excited states. In these excited states the dynamics of some methyl containing residues can also be studied.

NMRof peptides

Developments and applications of NMR spectroscopy especially with biomolecules has taken big strides over the decades. This review gives a brief overview of peptide analysis by NMR as carried out in the author’s laboratory. A brief introduction to peptide biomolecules and NMR useful parameters are discussed in the beginning. This is followed by diagnostics features observed in NMR for identification of secondary structures. It further goes on to show how a three dimensional structure could be obtained by all-important NOE and hydrogen bond information. Use of heteronuclear experiments, which could be done at natural abundance is also highlighted in getting more details of peptide structures.Applications using Solid state NMR at natural abundance in connecting peptide solution and x-ray structures is demonstrated with couple of examples.

Photocytotoxic organometallic compounds

Organometallic compounds have recently found applications in medicinal chemistry and as diagnostic tools in chemical biology. Naturally occurring biomolecules, viz., cobalamine, NiFe hydrogenase, Acetyl-CoA synthase, etc., also contain metal-carbon bonds. Among organometallic compounds having medicinal importance, (arene)ruthenium complexes, radioactive technetium complexes and ferrocene conjugates are notable ones. Applications of photoactive organometallic complexes or metal complexes conjugated with an organometallic moiety are of recent origin. Photodynamic therapy (PDT) is a promising method to treat cancer cells in presence of light. This review primarily focuses on different aspects of the chemistry of organometallic complexes showing photocytotoxic activities. Half-sandwich tungsten, iron or ruthenium complexes are known to show photonuclease and/or photo-crosslinking activity. Photoinduced organometallic CO releasing molecules also exert photocytotoxic activity. Attempts have been made in this review to highlight the photocytotoxic behavior of various metal complexes when conjugated with a photoactive organometallic moiety, viz., ferrocene.

From anomalies in neat liquid to structure, dynamics and function in the biological world

Water brings its remarkable thermodynamic and dynamic anomalies in the pure liquid state to biological world where water molecules face a multitude of additional interactions that frustrate its hydrogen bond network. Yet the water molecules participate and control enormous number of biological processes in manners which are yet to be understood at a molecular level. We discuss thermodynamics, structure, dynamics and properties of water around proteins and DNA, along with those in reverse micelles. We discuss the roles of water in enzyme kinetics, in drug-DNA intercalation and in kinetic-proof reading ( the theory of lack of errors in biosynthesis). We also discuss how water may play an important role in the natural selection of biomolecules. (C) 2011 Elsevier B. V. All rights reserved.

Photoresist functionalisation method for high-density protein microarrays using photolithography

Since the last decade, there is a growing need for patterned biomolecules for various applications ranging from diagnostic devices to enabling fundamental biological studies with high throughput. Protein arrays facilitate the study of protein-protein, protein-drug or protein-DNA interactions as well as highly multiplexed immunosensors based on antibody-antigen recognition. Protein microarrays are typically fabricated using piezoelectric inkjet printing with resolution limit of similar to 70-100 mu m limiting the array density. A considerable amount of research has been done on patterning biomolecules using customised biocompatible photoresists. Here, a simple photolithographic process for fabricating protein microarrays on a commercially available diazo-naphthoquinone-novolac-positive tone photoresist functionalised with 3-aminopropyltriethoxysilane is presented. The authors demonstrate that proteins immobilised using this procedure retain their activity and therefore form functional microarrays with the array density limited only by the resolution of lithography, which is more than an order of magnitude compared with inkjet printing. The process described here may be useful in the integration of conventional semiconductor manufacturing processes with biomaterials relevant for the creation of next-generation bio-chips.

Silver nanoparticles modified nanocapsules for ultrasonically activated drug delivery

Novel ultrasound-sensitive nanocapsules were designed via layer-by-layer assembly (LbL) of polyelectrolytes for remote activated release of biomolecules/drug. Nanocapsules embedded with silver nanoparticles in the walls were synthesized by alternate assembly of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on silica template followed by nanoparticle synthesis and subsequent template removal thus yielding nanocapsules. The silver NPs were synthesized in situ within the capsule walls under controlled conditions. The nanocapsules were found to be well dispersed and the silver NPs were evenly distributed within the shell. FITC-dextran permeated easily into the capsules containing silver NP's due to the pores generated during the formation of NP's. When the loaded nanocapsules were sonicated, the presence of the silver NPs in the shell structure led to rupturing of the shell into smaller fragments thus releasing the FITC-dextran. Such nanocapsules have the potential to be used as drug delivery vehicles and offer the scope for further development in the areas of modern medicine, material science, and biochemistry. (C) 2012 Elsevier B.V. All rights reserved.

MECHANISM OF CELL LYSIS IN MICROFLUIDIC CHANNEL WITH INTEGRATED NANOCOMPOSITE ELECTRODES

This paper reports on the characterization of an integrated micro-fluidic platform for controlled electrical lysis of biological cells and subsequent extraction of intracellular biomolecules. The proposed methodology is capable of high throughput electrical cell lysis facilitated by nano-composite coated electrodes. The nano-composites are synthesized using Carbon Nanotube and ZnO nanorod dispersion in polymer. Bacterial cells are used to demonstrate the lysis performance of these nanocomposite electrodes. Investigation of electrical lysis in the microchannel is carried out under different parameters, one with continuous DC application and the other under DC biased AC electric field. Lysis in DC field is dependent on optimal field strength and governed by the cell type. By introducing the AC electrical field, the electrokinetics is controlled to prevent cell clogging in the micro-channel and ensure uniform cell dispersion and lysis. Lysis mechanism is analyzed with time-resolved fluorescence imaging which reveal the time scale of electrical lysis and explain the dynamic behavior of GFP-expressing E. coli cells under the electric field induced by nanocomposite electrodes. The DNA and protein samples extracted after lysis are compared with those obtained from a conventional chemical lysis method by using a UV-Visible spectroscopy and fluorimetry. The paper also focuses on the mechanistic understanding of the nano-composite coating material and the film thickness on the leakage charge densities which lead to differential lysis efficiency.

Detection of Hepatitis B DNA Sequences on Polyelectrolyte Based Non-Covalently Functionalized Flexible Plastic Substrates

Development of simple functionalization methods to attach biomolecules such as proteins and DNA on inexpensive substrates is important for widespread use of low cost, disposable biosensors. Here, we describe a method based on polyelectrolyte multilayers to attach single stranded DNA molecules to conventional glass slides as well as a completely non-standard substrate, namely flexible plastic transparency sheets. We then use the functionalized transparency sheets to specifically detect single stranded Hepatitis B DNA sequences from samples. We also demonstrate a blocking method for reducing non-specific binding of target DNA sequences using negatively charged polyelectrolyte molecules. The polyelectrolyte based functionalization method, which relies on surface charge as opposed to covalent surface linkages, could be an attractive platform to develop assays on inexpensive substrates for low cost biosensing.

A fast NMR method for resonance assignments: application to metabolomics

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled (C-12) or C-13-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D C-13, H-1] HSQC-TOCSY, (2) 2D H-1-H-1 TOCSY and (3) 2D C-13-H-1 HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D C-13, H-1] HSQC-TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete H-1 and C-13 assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.