Exogenous chondroitin sulfate glycosaminoglycan associate with arginine-rich peptide–DNA complexes to alter their intracellular processing and gene delivery efficiency

AbstractArginine-rich peptides have been used extensively as efficient cellular transporters. However, gene delivery with such peptides requires development of strategies to improve their efficiency. We had earlier demonstrated that addition of small amounts of exogenous glycosaminoglycans (GAGs) like heparan sulfate or chondroitin sulfate to different arginine-rich peptide–DNA complexes (polyplexes) led to an increase in their gene delivery efficiency. This was possibly due to the formation of a ‘GAG coat’ on the polyplex surface through electrostatic interactions which improved their extracellular stability and subsequent cellular entry. In this report, we have attempted to elucidate the differences in intracellular processing of the chondroitin sulfate (CS)-coated polyplexes in comparison to the native polyplexes by using a combination of endocytic inhibitors and co-localization with endosomal markers in various cell lines. We observed that both the native and CS-coated polyplexes are internalized by multiple endocytic pathways although in some cell lines, the coated polyplexes are taken up primarily by caveolae mediated endocytosis. In addition, the CS-coat improves the endosomal escape of the polyplexes as compared to the native polyplexes. Interestingly, during these intracellular events, exogenous CS is retained with the polyplexes until their accumulation near the nucleus. Thus we show for the first time that exogenous GAGs in small amounts improve intracellular routing and nuclear accumulation of arginine-based polyplexes. Therefore, addition of exogenous GAGs is a promising strategy to enhance the transfection efficiency of cationic arginine-rich peptides in multiple cell types

Leishmania actin binds and nicks kDNA as well as inhibits decatenation activity of type II topoisomerase

Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling

Inhibition of Protein Aggregation: Supramolecular Assemblies of Arginine Hold the Key

BACKGROUND: Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. METHODOLOGY: We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Abeta(1-42)) peptide is modified. Changes in the hydrodynamic volume of Abeta(1-42) in the presence of arginine measured by size exclusion chromatography show that arginine binds to Abeta(1-42). Arginine increases the solubility of Abeta(1-42) peptide in aqueous medium. It decreases the aggregation of Abeta(1-42) as observed by atomic force microscopy. CONCLUSIONS: Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation

Inorganic particle synthesis via macro and microemulsions: a micrometer to nanometer landscape

"Nanotechnology" is now very well known as one of the most important key technologies in science and industry. In the field of material science and engineering, nanoparticles should be unit materials, as well as atoms and molecules, to build ceramics, devices, catalysts, and machines, and the "nanoparticle technology" is thus attracting. This novel technology includes various methodologies for nanoparticles: preparation, surface-modification via chemical and/or physical treatments, immobilization and arrangement on supports or substrates, to achieve high performance for luminescence properties in light emitting devices, and high efficiency for catalytic and photocatalytic reactions in chemical synthesis, chemical decomposition, and artificial photosynthesis, etc. It should be needless to say that the preparation of nanoparticles, having precisely controlled particle size, size distribution, chemical composition, and surface properties, is essentially important to realize "true nanoparticle technology". This book, written by Dr. Dibyendu Ganguli and Dr. Munia Ganguli, deals with the preparation methodologies for inorganic nanoparticles using macro- and microemulsions as "microreactor". There are several differences between these two emulsions, in addition to water droplet size: thermodynamic stability, and fusion-redispersion dynamics of the droplets. The properties of the nanoparticles prepared in these emulsion systems are seriously influenced and controlled by the selection of dynamic and static conditions

Studies on the Effect of Li2SO4 on the Structure of Lithium Borate Glasses

Thermal and spectroscopic investigations have been carried out on a number of glasses with a wide range of compositions in the pseudoternary glass system, Li2SO4-Li2O-B2O3, to understand the role of sulfate ions in modifying the borate glass structure. Both nuclear magnetic resonance (NMR) and infrared (IR) spectroscopic results indicate that four-coordinate boron atoms are retained in the glass structure to a greater extent in sulfate-containing glasses than in pure lithium borate glasses. There seems to be some evidence for the existence of sulfoborate-type units in Raman spectra in the region of 800-960 cm(-1). These conclusions are supported by the observed behavior of glass transition temperatures and molar volumes. The possibility of formation of sulfoborate-type units is discussed from bonding and thermodynamic points of view

Studies of ternary Li2SO4-Li2O-P2O5 glasses

Glasses in a wide range of compositions in the ternary system xLi(2)SO(4-y)Li(2)O-zP(2)O(5) where x ranges from 0 to 30 mol%, y ranges from 35 to 55 mol% and z ranges from 25 to 50 mol% have been prepared and their properties measured using infra-red, Raman, and P-31 magic angle spinning nuclear magnetic resonance spectroscopic techniques. We conclude that a random close packing of phosphate and sulphate ions which also leads to formation of connected voids in the structure is consistent with our data. There is also evidence for formation of condensed sulphate-phosphate species in the liquid which may be retained in the glass structure. (C) 1999 Elsevier Science B.V. All rights reserved

Structural role of PbO in Li<SUB>2</SUB>O-PbO-B<SUB>2</SUB>O<SUB>3</SUB> glasses

Thermal and spectroscopic studies have been carried out on a number of glasses with wide ranging compositions in the pseudoternary glass system Li2O-PbO-B2O3 in order to understand the structural role of PbO in these glasses. Infrared, Raman, and 11B MAS-NMR results indicate that PbO behaves as a network former in these glasses and is possibly incorporated in the network as [PbO4/2]2- units. The formation of [PbO4/2]2- units leads to the creation of neutral three-coordinated boron (B03) which, in turn, leads to the reformation of four-coordinated boron (B-4) in the structure at the expense of two-coordinated (B-2) and singly coordinated (B2-1) boron atoms. The variations of glass transition temperatures (Tg) and molar volumes also support this model

Frostbite – manifestation and mitigation

Frostbite is a severe health condition that is manifested in situations where an individual is exposed to extreme cold conditions. Touching materials such as ice packs/ dry ice, or just being exposed to a low wind speed in air temperature below −15 °C can result in a freeze-burn. The manifestations vary from a self-healing superficial burn (frostnip) on skin to conditions as severe as gangrenous necrosis, leading to auto-amputation of the affected limb. While ectotherms possess biological mechanisms to counter cold and can survive at sub-zero temperatures, humans depend mainly on the involuntary act of shivering or turn to insulating, protective gear for cold tolerance. For aiding frostbite patients, post-injury treatments are currently employed and prophylactic strategies are not commonly available. This review discusses frostbite manifestations – focusing on the therapies available and discusses various animal models used in frostbite research

Nanoparticles from cationic copolymer and DNA that are soluble and stable in common organic solvents

DNA by virtue of its superlative ability to self-assemble has found use beyond biological research in the design and fabrication of nanomaterials. However, developing novel DNA-based materials for chemical applications might be restricted due to the insoluble nature of DNA in most common organic solvents. In this Communication, we are reporting the first demonstration of making DNA soluble in a variety of nonbiological solvents such as acetonitrile, benzene, dimethyl sulfoxide (DMSO), and tetrahydrofuran with the help of poly(ethylene glycol) (PEG)-based cationic random copolymers. Because of complex formation between cationic copolymer and anionic DNA, nanoparticles are formed. These nanoparticles are expected to exhibit micelle-like structures with a nanometric core of cationic units neutralized by phosphate anions of DNA, surrounded by a shell of PEG segments. As PEG is soluble in the organic solvents used in this study, nanoparticles are stable in these solvents, making entrapped DNA soluble in these organic solvents

Complex formation between cationically modified gold nanoparticles and DNA: an atomic force microscopic study

Atomic force microscopy has been used for direct visualization of the wrapping of DNA around 30-nm-sized functionalized gold nanoparticles for the first time. The morphology of the complexes seems to be dictated by the relative concentration of the nanoparticles and DNA. A higher concentration of the former leads to the formation of a network of nanoparticles assembled on DNA. This assembly pattern seems to be significantly different from the manner in which cationically modified gold nanoparticles of smaller size (&#60;5 nm) arrange linearly on DNA, as shown in the literature. A DNA−gold nanoparticle can be developed as a model system for in vitro studies on the mechanism of DNA condensation and also for developing novel methods of nanoparticle self-assembly on the DNA template