Fabrication and Therapeutic Process of a Green Silver-Nanoparticle-Embedded Mucilage Microsphere for Pathogenic-Bacteria-Infected Second-Degree Burn and Excision Wounds.

Multidrug-resistant bacteria are a serious problem in biomedical applications that decrease the wound healing process and increase the mortality rate. Therefore, in this study, we have prepared a green-synthesized silver-nanoparticle-encapsulated mucilage microsphere (HMMS@GSNP) from Hibiscus rosa sinensis leaves and applied it to pathogen-infected burn and excision wounds. Biophysical properties like size, polydispersity index, absorbance capacity, and drug release were measured by different techniques like field-emission scanning electron microscopy, dynamic light scattering, swelling ratio, etc. The strong antibacterial activity of a HMMS@GSNP microsphere was measured by minimum inhibitory concentration assay, minimum bactericidal concentration assay, and agar well diffusion methods. The HMMS@GSNP microsphere enhanced the cell viability, cell proliferation, migration, antioxidant, and antiinflammation activity compared to untreated GSNP and HMMS, as quantified by MTT assay, BrdU assay, scratch wound assay, reactive oxygen species scavenging assay, and Western blot analysis, respectively. In the in vivo experiment, we used a methicillin-resistant Staphylococcus aureus bacteria-infected, burn-and-excision-wound-created male BALB/c mice model. The HMMS@GSNP-treated burn-and-excision-wound-infected mice showed significant results compared to other groups (untreated, Silverex Ionic Gel, AgNO3, HMMS, and GSNP), and the mice tissues were utilized for bacteria count, immunoblot analysis, histological studies, and real-time polymerase chain reaction. Thus, the HMM@GSNP microsphere is an excellent therapeutic material that can be used as a topical agent for the management of chronic wound therapy.

Facile process ofHibiscusmucilage polymer formulation usingHibiscus rosa-sinensisleaves to treat second-degree burn and excision wounds.

Mucilage is a sticky substance found in various plants and microorganisms and is made up of proteins and polysaccharides. Mucilage fromHibiscus rosa sinensisisis a complex polysaccharide traditionally used to treat different skin diseases. In our study, we fabricated mucilage polymer fromHibiscus rosa sinensisleaves and evaluated its potential application in second-degree burns and excision wounds. The physical properties of Hibiscus mucilage (HM) polymer were demonstrated by using Ultraviolet-visible absorption spectroscopy, x-ray diffraction, Fourier transform infrared spectroscopy, dynamic light scattering, Scanning electron microscopy, Brunauer-Emmett-Tellerand, Swelling ratio. The human cell lines WI-38, and HaCaT have been used forin-vitroexperiments like MTT, scratch wound, BrdU, ROS scavenging assays, and western blot analysis. The results of the MTT, scratch-wound, and BrdU assay indicated that the HM polymer is nontoxic in nature and also enhances both the properties of cellular migration and proliferation, respectively. On the other hand, the result of the ROS scavenging assay suggested that HM polymer enhances the antioxidant activity of cells while the western blot analysis designated that the HM polymer treatment caused downregulation of the pro-inflammatory cytokine IFN-γand upregulation of the pAkt (Serine 473) protein, and TGF-ß1 signaling pathway. Therefore, allin-vitroexperimental studies recommended that HM polymer is biocompatible and has antioxidant and anti-inflammatory effects. In thein vivoexperiment, second-degree burns and excision wounds were created on the dorsal surface of male BALB/c mice. After the sixth day of HM polymer treatment have developed new tissue, hair follicles, blood vessels,α-SMA, and Collagen type-1 fiber on the burn and excision wound area while the 11th day of HM polymer treatment cured the wound area significantly. Therefore, it could be contemplated that HM polymer is a potential agent for treating different wounds in the near future.

Antibiofilm potential of nanonized eugenol against Pseudomonas aeruginosa.

AIMS: The purpose of this study was to synthesize a nanoform of eugenol (an important phytochemical with various pharmacological potentials) and to investigate its antibiofilm efficacy on Pseudomonas aeruginosa biofilm. METHODS AND RESULTS: Colloidal suspension of eugenol-nanoparticles (ENPs) was synthesized by the simple ultrasonic cavitation method through the emulsification of hydrophobic eugenol into hydrophilic gelatin. Thus, the nanonization process made water-insoluble eugenol into water-soluble nano-eugenol, making the nanoform bioavailable. The size of the ENPs was 20-30 nm, entrapment efficiency of eugenol within gelatin was 80%, and release of eugenol from the gelatin cap was slow and sustained over 5 days. Concerning the clinically relevant pathogen P. aeruginosa, ENPs had higher antibiofilm (for both formation and eradication) activities than free eugenol. Minimal biofilm inhibitory concentration and minimal biofilm eradication concentration of ENP on P. aeruginosa biofilm were 2.0 and 4.0 mM, respectively. In addition, the measurement of P. aeruginosa biofilm biomass, biofilm thickness, amount of biofilm extra-polymeric substance, cell surface hydrophobicity, cell swarming and twitching efficiencies, cellular morphology, and biofilm formation in catheter demonstrated that the antibiofilm efficacy of nano-eugenol was 30%-40% higher than that of bulk eugenol. CONCLUSION: These results signify that future pharmacological and clinical studies are very much required to investigate whether ENPs can act as an effective drug against P. aeruginosa biofilm-mediated diseases. Thus, the problem of intrinsic antibiotic tolerance of biofilm-forming cells may be minimized by ENPs. Moreover, ENP may be used as a potential catheter-coating agent to inhibit pseudomonal colonization on catheter surfaces and, therefore, to reduce catheter-associated infections and complications.

A review on oligomeric polydispersity and oligomers-dependent holding chaperone activity of the small heat-shock protein IbpB of Escherichia coli.

Inclusion body-associated proteins IbpA and IbpB of MW 16 KDa are the two small heat-shock proteins (sHSPs) of Escherichia coli, and they have only holding, but not folding, chaperone activity. In vitro holdase activity of IbpB is more than that of IbpA, and in combination, they synergise. Both IbpA and IbpB monomers first form homodimers, which as building blocks subsequently oligomerize to make heavy oligomers with MW of MDa range; for IbpB, the MW range of heavy oligomers is 2.0-3.0 MDa, whereas for IbpA oligomers, the values in MDa are not so specified/reported. By temperature upshift, such large oligomers of IbpB, but not of IbpA, dissociate to make relatively small oligomeric assemblies of MW around 600-700KDa. The larger oligomers of IbpB are assumed to be inactive storage form, which on facing heat or oxidative stress dissociate into smaller oligomers of ATP-independent holding chaperone activity. These smaller oligomers bind with stress-induced partially denatured/unfolded and thereby going to be aggregated proteins, to give them protection against permanent damage and aggregation. On withdrawal of stress, IbpB transfers the bound substrate protein to the ATP-dependent bi-chaperone system DnaKJE-ClpB, having both holdase and foldase properties, to finally refold the protein. Of the two sHSPs IbpA and IbpB of E. coli, this review covers the recent advances in research on IbpB only.

Nanonization of a chemically synthesized flavone HMDF (3-hydroxy-3',4'-methylenedioxyflavone) by entrapping within calcium phosphate nanoparticles and exploring its antioxidant role on neural cellsin vitroand zebrafishin vivo.

The chemical synthesis of 3-hydroxy-3',4'-methylenedioxyflavone (HMDF) was reported to generate a modified flavone of potent antioxidant activity with significant neuropharmacological properties. In this study, HMDF was nanonized by entrapping within calcium phosphate nanoparticles (CPNPs). HMDF-CPNPs were of (i) size 25 nm, (ii) zeta potential (-) [22 ± 3] mV and (iii) entrapment efficiency 67%. HMDF-CPNPs, but not HMDF alone, inhibited thein vitroactivity of acetylcholinesterase enzymes to break down the major neurotransmitter compound acetylcholine. Moreover, nanonized HMDF had more antioxidant activity than bulk HMDF, as observed from its ability to protect mouse neural (N2A) cells from oxidative damage caused by H2O2exposure at the levels of cell viability, intracellular reactive oxygen species, mitochondrial membrane potential, cell cycle stages, nuclear integrity and neural connectivity. Anin vivostudy on zebrafish larvae (Denio rerio) also demonstrated that H2O2-mediated larval death was checked by HMDF-CPNP treatment. These results, therefore, suggest that HMDF-CPNPs may be developed as a potential antioxidant, particularly as a neuroprotectant.

Nano-Antibacterials Using Medicinal Plant Components: An Overview.

Gradual emergence of new bacterial strains, resistant to one or more antibiotics, necessitates development of new antibacterials to prevent us from newly evolved disease-causing, drug-resistant, pathogenic bacteria. Different inorganic and organic compounds have been synthesized as antibacterials, but with the problem of toxicity. Other alternatives of using green products, i.e., the medicinal plant extracts with biocompatible and potent antibacterial characteristics, also had limitation because of their low aqueous solubility and therefore less bioavailability. Use of nanotechnological strategy appears to be a savior, where phytochemicals are nanonized through encapsulation or entrapment within inorganic or organic hydrophilic capping agents. Nanonization of such products not only makes them water soluble but also helps to attain high surface to volume ratio and therefore high reaction area of the nanonized products with better therapeutic potential, over that of the equivalent amount of raw bulk products. Medicinal plant extracts, whose prime components are flavonoids, alkaloids, terpenoids, polyphenolic compounds, and essential oils, are in one hand nanonized (capped and stabilized) by polymers, lipids, or clay materials for developing nanodrugs; on the other hand, high antioxidant activity of those plant extracts is also used to reduce various metal salts to produce metallic nanoparticles. In this review, five medicinal plants, viz., tulsi (Ocimum sanctum), turmeric (Curcuma longa), aloe vera (Aloe vera), oregano (Oregano vulgare), and eucalyptus (Eucalyptus globulus), with promising antibacterial potential and the nanoformulations associated with the plants' crude extracts and their respective major components (eugenol, curcumin, anthraquinone, carvacrol, eucalyptus oil) have been discussed with respect to their antibacterial potency.

Surface Modification by Media Organics Reduces the Bacterio-toxicity of Cupric Oxide Nanoparticle against Escherichia coli.

Prevalence of antibiotic-resistant bacteria demands alternatives to antibiotics. Copper-based nanoparticles with a high antibacterial property may be a solution to the problem. It is, therefore, important to understand the mode of antibacterial action of the nanoparticles (NPs). Despite reports on induction of reactive oxygen species (ROS) in bacteria by copper and copper-oxide nanoparticles and involvement of such ROS in cell killing, it is still unclear (a) if surface modification of the nanoparticles by media organics has any role on their antibacterial potency and (b) whether the bactericidal effects of these NPs are 'particle-specific' or 'ion-specific' in nature. We address these issues for cupric oxide nanoparticle (CuO-NP) in this study. Instead of nutrient medium, when E. coli bacterial cells were suspended in saline (0.9% NaCl), CuO-NP had a more anti-bacterial effect, with MBC (minimum bactericidal concentration) value of 6 µg/mL, than in nutrient medium with MBC value of 160 µg/mL. Moreover, the lysine-modified CuO-NP in saline had MBC at 130 µg/mL. Thus, unmodified CuO-NP was more efficient killer than modified one. Our finding further revealed that in saline;CuO-NP had 'particle-specific' antibacterial effect through generation of ROS and consequent oxidative damage by lipid peroxidation, protein oxidation and DNA degradation in cells.

Nanonized tetracycline cures deadly diarrheal disease 'shigellosis' in mice, caused by multidrug-resistant Shigella flexneri 2a bacterial infection.

We reported earlier about nano-formulation of tetracycline through its entrapment within calcium-phosphate nano-particle (CPNP) and about killing of pathogenic bacterium Shigella flexnari 2a, resistant to tetracycline (and 9 other antibiotics), by the nanonized antibiotic (Tet-CPNP). Here, we report on therapeutic role of Tet-CPNP against deadly diarrheal disease 'shigellosis' in mice, caused by Shigella infection. Our findings revealed that occurrence of mushy-stool excretion, colon-length shortening, weight-loss and bacterial colonization in gastrointestinal tract of mice due to shigellosis was significantly reduced by Tet-CPNP treatment. Histo- and immuno-logical studies showed that changes in morphology and level of inflammatory cytokines TNF-α, IL-1ß and IFN-γ in intestinal tissue of Shigella-infected mice were reverted to almost normal features by Tet-CPNP treatment. Bulk tetracycline had no anti-shigellosis action. Thus, nanonization of tetracycline rejuvenated the old, cheap, broad-spectrum antibiotic from obsolescence (due to resistance generation), making it highly beneficial for diarrhea-prone developing countries with limited health-care budgets.

Antibacterial and Photocatalytic Properties of ZnO-9-Aminoacridine Hydrochloride Hydrate Drug Nanoconjugates.

The development of nanomaterial-based hybrid systems for healthcare and energy-related materials has attracted significant attention nowadays. Here, we have designed a nanocomposite of ZnO nanoparticles (NPs) with anticancer therapeutic drug 9-aminoacridine hydrochloride hydrate (9AA-HCl) for antibacterial and photocatalytic activities. Spectroscopic studies reveal that the photoinduced electron transfer from photoexcited 9AA-HCl to the conduction band of ZnO NP causes the generation of the reactive oxygen species (ROS), which is responsible for antibacterial activity and photocatalytic properties. It is seen that the efficiency of photodegradation of dye molecules increases in ZnO-9AA-HCl nanoconjugated systems than pure ZnO nanoparticles because of efficient charge separation. In addition, the antibacterial efficacy of the nanoconjugate is investigated using a strain of Gram-negative bacteria where the cell-killing activities are observed 99.99 and 100% for 20 and 21 µL/mL nanoconjugate, respectively, and very little cell-killing activity is observed for free ZnO NPs and free drug. Moreover, it is also observed that the nanoconjugate generates sufficient intracellular ROS that can hydrolyze 2',7'-dichlorodihydrofluoresceindiacetate (DCFH-DA) to highly fluorescent 2',7'-dichlorofluorescein (DCF). The outcome of the study will provide valuable information for designing new-edge nanoconjugate materials for potential applications in photocatalytic and antibacterial activities.

A simple robust method of synthesis of copper-silver core-shell nano-particle: evaluation of its structural and chemical properties with anticancer potency.

A simple method of synthesis of a stable bimetallic copper-silver nano-particle (CuAg-NP) was developed by successive reduction of Cu(NO3)2 and AgNO3, using hydrazine hydrate as the reducing agent and gelatin and poly-vinyl pyrrolidone (PVP) as the capping agents. The round-shaped particles were of a core-shell structure with a core of Cu0 atoms surrounded by a shell of Ag0 atoms. The size and the mol. wt. of the NPs were (100 ± 10) nm and (820 ± 157) Kd, respectively; the particles were crystalline in nature and 90% of the precursors Cu(NO3)2 and AgNO3 were converted to the NPs. The particles were more toxic to cancer cells than normal cells; the dose of the NPs (4-5 µg ml-1), that killed about 75% of the different human cancer cell lines viz, HepG2 (liver cancer), A549 (lung cancer) and AGS (stomach cancer), killed only about 22.5% of the normal cell lines viz, WRL68 (liver) and WI38 (lung). Therefore, the NP may be developed as a potent anticancer drug in future. The more detailed study on the cytotoxicity of the CuAg-NP on the HepG2 cell line revealed that the particles caused cell cycle arrest in a G2/M phase, depolarization of mitochondrial membrane potential, translocation of phosphatidylserine residues from inner to outer leaflets of cell membrane and DNA degradation; these phenomena confirmed that the NP-induced cell death was apoptotic in nature.

Calcium phosphate-quercetin nanocomposite (CPQN): A multi-functional nanoparticle having pH indicating, highly fluorescent and anti-oxidant properties.

Calcium phosphate quercetin nanocomposite (CPQN) i.e., quercetin entrapped in calcium phosphate nanoparticle was synthesized by a precipitation method at 80°C, taking ammonium hydrogen phosphate, calcium nitrate and quercetin as precursors and sodium citrate as stabilizer. The nanocomposite suspension had different color at different pH values, a property that could render the nanoparticle a pH indicator. Besides color, the particles also had different size, shape, stability and quercetin content with change of pH. In addition, the CPQN was highly fluorescent having two sharp emission peaks at 460 and 497nm, when excited at 370nm; by this property it behaved as an effective fluorophore to label biological cell. Moreover, the nanocomposite had potential anti-oxidant property, for which mortality of mouse neuroblastoma cell N2A, by H2O2-induced oxidative stress, was found to be lowered by the pre-treatment of the cells with CPQN.

Metallic copper nanoparticles induce apoptosis in a human skin melanoma A-375 cell line.

In two earlier communications (Chatterjee et al 2012 Nanotechnology 23 085103, Chatterjee et al 2014 Nanotechnology 25 135101), we reported the development of a simple and unique method of synthesizing highly stable metallic copper nanoparticles (Cu NPs) with high antibacterial activity. Here we report on the cytotoxic potency of the NPs against cancer cells. The value of the IC50 dose of the Cu NPs against human skin cancer cell A-375 was found to be 1.71 µg ml-1 only, which was much less than values reported so far, and this concentration had no cytotoxic effect on normal white blood cells. The NPs caused (i) lowering of cell membrane rigidity, (ii) DNA degradation, (iii) chromosomal condensation, (iv) cell cycle arrest in the G2/M phase, (v) depolarization of the mitochondrial membrane and (vi) apoptosis of cells. Cellular apoptosis occurred in the caspase-9-mediated intrinsic pathway. This study revealed that our Cu NPs had high anticancer properties by killing tumor cells through the apoptotic pathway. Since this particle has high antibacterial activity, our Cu NPs might be developed in future as a dual action drug-anticancer as well as antibacterial.

Green synthesized cerium oxide nanoparticle: A prospective drug against oxidative harm.

Cerium oxide nanoparticle (CeONP) of size 2-3nm was synthesized by a new, simple and green method at ambient temperature, using cerium nitrate as prime precursor and Aloe vera leaf extract as stabilizing agent. Of the two oxidation states (+3) and (+4) of cerium, it was dominantly present in (+3) state in CeONP and cyclic conversion of Ce(III)O→Ce(IV)O→Ce(III)O by reaction with H2O2 implied uninterrupted antioxidant property of CeONP. Moreover, the higher oxygen defect in the crystal lattice produced particles with higher antioxidant activity. CeONP was found to neutralize the deleterious effects of H2O2 viz., cell death, generation of intracellular reactive oxygen species and loss of connectivity in mouse neural cells. Therefore, CeONP might have potential use in future as an anti-oxidant drug.

Tetracycline-loaded calcium phosphate nanoparticle (Tet-CPNP): Rejuvenation of an obsolete antibiotic to further action.

BACKGROUND: Increasing resistance in bacteria towards antibiotics has made it imperative to research on their revitalization to combat infectious diseases. This study dealt with synthesis of a nano-form of the antibiotic tetracycline, its characterization and potency of killing different multi-drug resistant diarrhea-causing bacteria. METHODS: Nano-formulation was done by loading tetracycline within biocompatible calcium phosphate nanoparticle. The synthesized tetracycline-loaded calcium phosphate nanoparticle (Tet-CPNP) was characterized by the techniques like TEM, DLS, EDS, FTIR, spectrofluorimetry and dialysis. Bactericidal activity of nano-particulate tetracycline was investigated by agar plating, spectrophotometry, phase contrast-fluorescence-atomic force microscopy and flow cytometry techniques. RESULTS: The Tet-CPNPs were 8±5nm in size and nearly spherical in shape, efficiency of tetracycline loading in CPNP was about 20% and the release of antibiotic from Tet-CPNPs was sustainable during 7days. Minimum inhibitory concentration (MIC) of Tet-CPNP on multiple antibiotic (including tetracycline) resistant bacteria like Escherichia coli, Salmonella kentuckey and Shigella flexneri was in the range of 20-40µg/ml, whereas MIC of free tetracycline was in the range of 150-180µg/ml. NP-mediated cell filamentation and cell membrane disintegration caused cell killing. Moreover, death of Shigella-infected Zebra fish larvae was stalled by Tet-CPNP treatment. CPNP itself had no toxic effect on bacteria as well as on Zebra fish. CONCLUSION: Our nano-formulation of tetracycline might reclaim a nearly obsolete antibiotic to further potential function. GENERAL SIGNIFICANCE: Such a study on revival of an old, cheap, broad-spectrum antibiotic to further action is highly beneficial to developing countries with limited health care budgets.

GroEL to DnaK chaperone network behind the stability modulation of σ(32) at physiological temperature in Escherichia coli.

The stability of heat-shock transcription factor σ(32) in Escherichia coli has long been known to be modulated only by its own transcribed chaperone DnaK. Very few reports suggest a role for another heat-shock chaperone, GroEL, for maintenance of cellular σ(32) level. The present study demonstrates in vivo physical association between GroEL and σ(32) in E. coli at physiological temperature. This study further reveals that neither DnaK nor GroEL singly can modulate σ(32) stability in vivo; there is an ordered network between them, where GroEL acts upstream of DnaK.

A simple, fast and cost-effective method of synthesis of cupric oxide nanoparticle with promising antibacterial potency: Unraveling the biological and chemical modes of action.

BACKGROUND: Gradual attainment of bacterial resistance to antibiotics led us to develop a robust method of synthesis of stable, colloidal cupric oxide nanoparticle of physiological pH with potential antibacterial action. METHODS: Cu(II) oxide NP was synthesized by reduction-oxidation of CuCl2, using polyvinyl alcohol as stabilizer. Characteristics and antibacterial activity of the particles were investigated by techniques like UV-Vis spectrophotometry, DLS, AFM, TEM, EDS, FTIR, AAS, agar plating, FACS, gel electrophoresis and XPS. RESULTS: The NPs were about 50 nm in size and cubic in shape with two surface plasmon peaks at 266 and 370 nm and had semi-conducting behavior with a band gap of 3.40 and 3.96 eV. About 80% of precursor CuCl2 was converted to NP. The minimum inhibitory and the minimum bactericidal concentrations of CuO-NP were respectively 120 and 160 µg/mL for Escherichia coli and 180 and 195 µg/mL for Staphylococcus aureus in Luria-Bertani medium. In growth media, the NPs got modified by media organics with displacement of the stabilizer PVA molecules. This modified NP (around 240 nm) killed cells by generating ROS, which finally caused membrane lipid per-oxidation and chromosomal DNA degradation in NP-treated cells. CONCLUSION: Reports indicate that we are among the few who had prepared CuO-NP in colloidal form. The antibacterial potency of our particle in growth media was much promising than other reports. Our findings demonstrated that 'particle-specific' effect, not 'ion-specific' one, was responsible for the NP action. GENERAL SIGNIFICANCE: The NP may be used as a sterilizing agent in various bioprocesses and as substituent of antibiotics, after thorough toxicological study.

Mechanism of antibacterial activity of copper nanoparticles.

In a previous communication, we reported a new method of synthesis of stable metallic copper nanoparticles (Cu-NPs), which had high potency for bacterial cell filamentation and cell killing. The present study deals with the mechanism of filament formation and antibacterial roles of Cu-NPs in E. coli cells. Our results demonstrate that NP-mediated dissipation of cell membrane potential was the probable reason for the formation of cell filaments. On the other hand, Cu-NPs were found to cause multiple toxic effects such as generation of reactive oxygen species, lipid peroxidation, protein oxidation and DNA degradation in E. coli cells. In vitro interaction between plasmid pUC19 DNA and Cu-NPs showed that the degradation of DNA was highly inhibited in the presence of the divalent metal ion chelator EDTA, which indicated a positive role of Cu(2+) ions in the degradation process. Moreover, the fast destabilization, i.e. the reduction in size, of NPs in the presence of EDTA led us to propose that the nascent Cu ions liberated from the NP surface were responsible for higher reactivity of the Cu-NPs than the equivalent amount of its precursor CuCl2; the nascent ions were generated from the oxidation of metallic NPs when they were in the vicinity of agents, namely cells, biomolecules or medium components, to be reduced simultaneously.

Calcium phosphate nanoparticles: a study of their synthesis, characterization and mode of interaction with salmon testis DNA.

Calcium phosphate nanoparticles (CPNPs) are presently emerging as a second generation vector for efficient delivery and stabilization of nucleic acids inside cells, although the detailed mode of interaction between CPNPs and DNA is still obscure. This study discloses some features of the interaction. For this study, we synthesized CPNPs by a modified co-precipitation method and characterized the particles by different techniques such as dynamic light scattering, X-ray diffraction, electron dispersive spectroscopy, Fourier transform infra-red spectroscopy, differential thermal and thermo-gravimetric analysis, and atomic force, scanning and transmission electron microscopy. The characterization studies showed that the nanoparticles were spherical in shape, about 45 nm in size and were composed of the hydroxyapatite form of calcium phosphate; almost 90% of the starting materials were converted to nanoparticles (NPs). The different aspects of the interaction between CPNPs and salmon testis DNA were investigated using techniques such as UV-Vis spectrophotometry, circular dichroism, Fourier transform infra-red spectrometry, thermal denaturation, microviscometry, agarose gel electrophoresis, cyclic voltammetry and atomic force microscopy. The results revealed that CPNPs interacted with DNA with ~1 : 3.3 stoichiometry with a binding constant of the order of 10(4) M(-1) through groove-interacting mode and a single nanoparticle covered about 6.2 base pairs of the DNA chain. Moreover, the binding interaction was spontaneous, cooperative, exothermic and enthalpy-driven and some electrostatic nature of the binding was also evident; however, the non-polyelectrolyte contribution was dominant. The binding interaction finally caused an increase in the melting temperature of DNA from 70.8 °C to 75 °C and alteration of its secondary structure from the naturally occurring B-form to C-form.

In vitro holdase activity of E. coli small heat-shock proteins IbpA, IbpB and IbpAB: a biophysical study with some unconventional techniques.

E. coli small heat shock proteins IbpA and IbpB (inclusion body binding proteins A and B) are known to act as holding chaperones on denaturing, aggregate-prone proteins. But, there is no clear understanding about which of the IbpA and IbpB has more holdase activity and how the holdase activity of one was influenced by the presence of the other. This study was conducted to resolve the questions, using some uncommon physical techniques like dynamic light scattering, micro-viscometry and atomic force microscopy in addition to the common techniques of spectrophotometry and spectrofluorimetry. The holdase activity was investigated on the heat-denatured L-lactate dehydrogenase (LDH) of rabbit muscle. LDH was found to be deactivated completely without any aggregation at 52°C and with transient aggregation at 60°C; molecular dynamics simulation also revealed that at 52°C, denaturation occurred only at the active site of LDH. When LDH was allowed to be deactivated in the presence of IbpA, IbpB or (IbpA + IbpB), partial inhibition of i) denaturation at 52°C and ii) aggregation at 60°C were observed. The results further demonstrated that the holdase activity of IbpB was higher than that of IbpA and their combined effect was higher than their individual one.

Calcium chloride made E. coli competent for uptake of extraneous DNA through overproduction of OmpC protein.

In the standard method of transformation of Escherichia coli with extraneous DNA, cells are made competent for DNA uptake by incubating in ice-cold 100 mM CaCl(2). Analysis of the whole protein profile of CaCl(2)-treated E. coli cells by the techniques of one- and two-dimensional gel electrophoresis, MALDI-MS and immunoprecipitation revealed overproduction of outer membrane proteins OmpC, OmpA and heat-shock protein GroEL. In parity, transformation efficiency of E. coli ompC mutant by plasmid pUC19 DNA was found to be about 40 % lower than that of the wild type strain. Moreover, in E. coli cells containing groEL-bearing plasmid, induction of GroEL caused simultaneous overproduction of OmpC. On the other hand, less OmpC was synthesized in E. coli groEL mutant compared to its wild type counterpart, by CaCl(2)-shock. From these results it can be suggested that in the process of CaCl(2)-mediated generation of competence, the heat-shock chaperone GroEL has specific role in DNA entry into the cell, possibly through the overproduced OmpC and OmpA porins.