Article

Spectroscopic studies on the binding interaction of phenothiazinium dyes, azure A and azure B to double stranded RNA polynucleotides

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

This manuscript presents spectroscopic characterization of the interaction of two phenothiazinium dyes, azure A and azure B with double stranded (ds) ribonucleic acids, poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C). Absorbance and fluorescence studies revealed that these dyes bind to the RNAs with binding affinities of the order 10(6)M(-1) to poly(A).poly(U), and 10(5)M(-1) to poly(C).poly(G) and poly(I).poly(C), respectively. Fluorescence quenching and viscosity data gave conclusive evidence for the intercalation of the dyes to these RNA duplexes. Circular dichroism results suggested that the conformation of the RNAs was perturbed on interaction and the dyes acquired strong induced optical activity on binding. Azure B bound to all the three RNAs stronger than azure A and the binding affinity varied as poly(A).poly(U)>poly(C).poly(G)>poly(I).poly(C) for both dyes. Copyright © 2015. Published by Elsevier B.V.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... (U)], polycytidylic-polyguanylic acid [poly(C)·poly(G)] and polyinosinic-polycytidylic acid [poly(I).poly(C)] [3,[12][13][14][15][16][17][18][19]. ...
... By plotting 1/ absorbance intensity with respect to 1/ [RNA], the Benesi- Hildebrand association constant for the complex formation (K BH ) was derived from the ratio of the slope/intercept [31]. Several scenarios of such binding outcomes have been observed previously also [3,10,11,13,14,17,18]. ...
... Furthermore, negative band attributed to helicity registered no appreciable change. Both the dyes registered visible isoelliptic point at 288 nm with AU and at 270 nm with IC polynucleotide which recommends that the structural changes may be interdependent [18]. The reduction of CD signal at 268 nm may correspond to a minor change in number of base pair per turn in RNA helix [49]. ...
... These PHZ dyes have the skill to inactivate several forms of pathogenic agents in fresh plasma due to their more redox potential and their ability to effectively co-operate with visible light [14]. AZA (Scheme 1(a)) is a blue colored, synthetic cationic dye belonging to the PHZ group [15]. It is an essential dye able with different biological properties that potentiates its usage as a therapeutic agent [15]. ...
... AZA (Scheme 1(a)) is a blue colored, synthetic cationic dye belonging to the PHZ group [15]. It is an essential dye able with different biological properties that potentiates its usage as a therapeutic agent [15]. AZA is also known as an asymmetrical dimethylthionine has been studied for photo induced chemotherapeutic properties against carcinomas [16]. ...
... 69 Dyes like Azure B found to be affecting helical structure of DNA 67,72 and duplex RNA. 73 Whereas at cytotoxic level, they inhibition enzymes (monoamine oxidase A) (MAO-A), 74 involved in nervous system 75 related to human behavior. 76 Similarly, inhibition of glutathione reductase 77 disturbed redox homeostasis cellular. ...
Article
Full-text available
Globally, textile dyeing and manufacturing are one of the largest industrial units releasing huge amount of wastewater (WW) with refractory compounds such as dyes and pigments. Currently, wastewater treatment has been viewed as an industrial opportunity for rejuvenating fresh water resources and it is highly required in water stressed countries. This comprehensive review highlights an overall concept and in-depth knowledge on integrated, cost-effective cross-disciplinary solutions for domestic and industrial (textile dyes) WW and for harnessing renewable energy. This basic concept entails parallel or sequential modes of treating two chemically different WW i.e., domestic and industrial in the same system. In this case, contemporary advancement in MFC/MEC (METs) based systems towards Microbial-Electro-Fenton Technology (MEFT) revealed a substantial emerging scope and opportunity. Principally the said technology is based upon previously established anaerobic digestion and electro-chemical (photo/UV/ Fenton) processes in the disciplines of microbial biotechnology and electro-chemistry. It holds an added advantage to all previously establish technologies in terms of treatment and energy efficiency, minimal toxicity and sludge waste, and environmental sustainable. This review typically described different dyes and their ultimate fate in environment and recently developed hierarchy of MEFS. It revealed detail mechanisms and degradation rate of dyes typically in cathodic Fenton system under batch and continuous modes of different MEF reactors. Moreover, it described cost-effectiveness of the said technology in terms of energy budget (production and consumption), and the limitations related to reactor fabrication cost and design for future upgradation to large scale application.
... The azo containing dyes, most commonly used textile dyes, have reported mutagenic and carcinogenic effect on human being. There are certain dyes having ability to interact with the human DNA, once enter into the body through food chain and alter its function (Haq and Raj 2018;Khan and Kumar 2016). Dyes released through effluent enhance the biological oxygen demand (BOD) and chemical oxygen demand (COD) of the water bodies, decrease their visibility and affect the aquatic life badly (Vikrant et al. 2018). ...
... Phenoxazinium derivatives exhibit distinguished properties of longwave length absorption and emission properties, exceptional stability against photo-bleaching, and low toxicity [25][26][27][28], and they have recently played an important role in analysis attributing to these unique properties [29][30][31]. However, the effects of pH conditions on phenoxazinium type fluorophores themselves have been studied rarely, their pHsensitive optical properties and the (TD) DFT calculations are still vital and interesting for practical and scientific purposes. ...
Article
Full-text available
Oxazinium derivatives have recently played an important role in bioanalysis attributing to the distinguished properties, thus a detailed study of the structure-property relationship is especially significant. Herein, pH-sensitive optical properties of Nile Blue (1a), N-monoalkyl-Nile Blue (1b) and Azure A (1c) have been carried out in extreme acid and base conditions. Dyes 1a and 1c showed colorimetric changes by the protonation of nitrogen atom in strong acidic condition (pH < 2.0), and dyes 1a − c exhibited colorimetric changes by equilibrium between amino and imide groups in very strong basic case (pH > 7.6). Besides, their fluorescent properties were closed to ON − OFF and OFF − ON emissions at 640–820 nm under strong acidic and basic conditions. Moreover, the absorption and emission properties were reversible, and there were no remarkable optical intensity changes of dyes 1a − c under subacidic and neutral solutions (pH = 3.0–7.0). The (TD) DFT calculations were used to optimize the most stable structures of their corresponding protonated and deprotonated forms, and their absorption and emission properties were also explained. Their fluorescent properties nearly ON-OFF and OFF − ON in strong acidic and basic conditions at near-infrared region will give the possible application in pH detection for extreme conditions. Graphical abstractᅟ
... The mass spectral peaks at approximately m=z = 284 are assigned to the parent MB, and those at m=z = 285, 286, and 287 can be assigned to the hydrogenated MB denoted as MB:nH. The mass spectral peaks at approximately m=z = 270 are assigned to azure B (AB), the molecular structure of which is depicted in Fig. 4. 35) The mass spectral peaks at m=z = 271, 272, and 273 can be assigned to hydrogenated AB denoted as AB:nH. The formation of AB will be discussed later because AB, which is not intentionally mixed in the nontreated aqueous solution, is not what we expect in the nontreated aqueous solution. ...
Article
We have performed matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) on methylene-blue aqueous solutions treated with three-dimensionally integrated micro-solution plasma, in which we have acquired the time evolution of mass spectra as a function of treatment time. The time evolution of mass spectral peak intensities for major detected species has clearly indicated that the parent methylene-blue molecules are degraded through consecutive reactions. The primary reaction is the oxidation of the parent molecules. The oxidized species still have two benzene rings in the parent molecules. The secondary reactions are the separation of the oxidized species and the formation of compounds with one benzene ring. We have also performed the numerical fitting of the time evolution of the mass spectral peak intensities, the results of which have indicated that we must assume additional primary reactions before the primary oxidation for better agreement with experimental results.
... Some dyes reveal mutagenic potentiality (Hunger, 2003). One of them is Azure-B, widely used in the textile industry, which is able to intercalate with the helical structure of DNA (Christie, 2007;Haq & Raj, 2018) and duplex RNA (Khan & Kumar, 2016), as it can be partitioned to the lipid membrane of the cells (Li, Zhang, Tang, Zhang, & Mao, 2014). It is noteworthy that this dye can reveal cytotoxic effects by acting as a notable reversible inhibitor of monoamine oxidase A (MAO-A), according to in vitro tests (Petzer, Harvey, Wegener, & Petzer, 2012), which is an intracellular enzyme of the nervous system central (Factor and Weiner, 2007) that plays an important role in human behavior (Di Giovanni et al., 2008). ...
Article
Full-text available
The water is an essential resource for life on the planet and for human development. The textile industry is one of the anthropogenic activities that most consume water and pollute water bodies. Therefore, the present work aims to undertake a review on the main effects of the release of industrial dyes and the essential bioremediation mechanisms. The textile dyes significantly compromise the aesthetic quality of water bodies, increase biochemical and chemical oxygen demand (BOD and COD), impair photosynthesis, inhibit plant growth, enter the food chain, provide recalcitrance and bioaccumulation, and may promote toxicity, mutagenicity and carcinogenicity. In spite of this, the bioremediation of textile dyes, that is, the transformation or mineralization of these contaminants by the enzymatic action of plant, bacteria, extremophiles and fungi biomasses is fully possible. Another option is the adsorption. Despite some disadvantages, the bioremediation is essentially positive and can be progressively enhanced by modern biotechnological techniques that are related to the generation of more degrading and more resistant engineered organisms. This is a sustainable solution that provides a fundamental and innovative contribution to conventional physicochemical treatments. The resources of environmental biotechnology can, therefore, be used as tangible technological solutions for the treatment of textile dye effluents and are related to the ethical imperative of ensuring the minimum necessary for a quality life for the humankind. Keywords: Phytoremediation, Effluents, Ecotoxicity, Mutagenicity, Carcinogenicity, Microorganisms
... This direct interaction of thionine with mRNA is confirmed by circular dichroism (CD) induced in the thionine absorption bands attributed to intimate contact of the achiral dye with the chiral mRNA molecule. Intercalation and electrostatic (outside) binding modes have been distinguished previously for phenothiazinium dyes binding to DNA (22) and RNA (23). The optical signatures of Fig. 1 b are consistent with the RNA/dye ratio-dependent signatures of these binding modes reported for transfer RNA (24). ...
Article
Full-text available
Understanding the structure of messenger RNA lipid nanoparticles (mRNA-LNP) and specifically the microenvironment of the mRNA molecules within these entities is fundamental to advancing their biomedical potential. Here we show that a permeating cationic dye, thionine, can serve as a cryogenic electron microscopy (cryo-EM) contrasting agent by binding selectively to encapsulated mRNA without disturbing LNP morphology. Cryo-EM images identify the mRNA location revealing that mRNA may exist within solvent-filled cavities or may be substantially lipid-associated.
... Some of the synthetic dyes have genotoxic effects (Christie, 2007;Tiwari et al., 2016). For example, Azure-B dye interacts with DNA and RNA and cause mutagenic disorders (Haq et al., 2018;Khan and Suresh Kumar, 2016). Table 2 briefly describes the applications and toxic effects of some of the synthetic dyes. ...
Article
The total annual output of synthetic dyes exceeds 7 × 105 tons. About 1,000 tons of non-biodegradable synthetic dyes are released every year into the natural streams and water sources from textile wastes. The release of these colored wastewater exerts negative impact on aquatic ecology and human beings because of the poisonous and carcinogenic repercussions of dyes involved in coloration production. Therefore, with a growing interest in the environment, efficient technologies need to be developed to eliminate dyes from local and industrial wastewater. Supercritical water oxidation as a promising wastewater treatment technology has many advantages, such as a rapid reaction and pollution-free products. However, due to corrosion, salt precipitation and operational problems, supercritical water oxidation process did not gain expected industrial development. These technical difficulties can be overcome by application of non-corrosive subcritical water as a reaction medium. This work summarizes the negative impacts of dyes and role of subcritical and supercritical water and their efficiencies in dye oxidation processes.
... AZB is used in the staining of DNA, blood products, epithelial tissues, proteins, viruses, lignin, melanin, keratin fibers and malaria-infected cells [5]. It is also used in the diagnosis of amyloid accumulation related diseases [6], malignant melanoma, detection of oral cancer, oral cavity infection and neuro-degenerative diseases, etc., [7][8][9]. ...
... Besides, due to their high aromaticity, most of these compounds exhibit mutagenic and carcinogenic properties. In addition, these molecules are capable of intercalating with the duplex RNA (Khan and Kumar, 2016) and helical structure of DNA (Haq et al., 2018), thereby destroying the transcription processes of the cell. The majority of dye compounds are considered to be persistent organic pollutants (POPs) and may bio-accumulate in numerous species. ...
Article
The indispensable role of plastic products in our daily life is highlighted by the COVID-19 pandemic again. Disposable face masks, made of polymer materials, as effective and cheap personal protective equipment (PPE), have been extensively used by the public to slow down the viral transmission. The repercussions of this have generated million tons of plastic waste being littered into the environment because of the improper disposal and mismanagement amid. And plastic waste can release microplastics (MPs) with the help of physical, chemical and biological processes, which is placing a huge MPs contamination burden on the ecosystem. In this work, the knowledge regarding to the combined effects of MPs and pollutants from the release of face masks and the impacts of wasted face masks and MPs on the environment (terrestrial and aquatic ecosystem) was systematically discussed. In view of these, some green technologies were put forward to reduce the amounts of discarded face masks in the environment, therefore minimizing MPs pollution at its source. Moreover, some recommendations for future research directions were proposed based on the remaining knowledge gaps. In a word, MPs pollution linked to face masks should be a focus worldwide.
... This untreated or semi-treated effluent with high turbidity and color restrains the penetration of the oxygen and sunlight in the water bodies in which they are discharged causing destruction to the aquatic life (Crini 2006;Yaseen and Scholz 2019). Furthermore, textile dyes are known to cause various health hazards like dermatitis (Khan and Malik 2018), inactivation of the enzymatic activities (Copaciu et al. 2013), skin and eye irritation (Clark 2011), allergy (Sivarajasekar and Baskar 2014;Lellis et al. 2019), genotoxicity (Tiwari et al. 2016), intercalates DNA/RNA (Khan and Kumar 2016;Haq and Raj 2018), damages central nervous system (Petzer et al. 2012;Khan and Malik 2018), heapatoma , random mutations (Lacasse and Baumann 2012;Sivarajasekar and Baskar 2014;Duarte et al. 2016;Mani and Bharagava 2016;Piatkowska et al. 2018), cytotoxic (Ferraz et al. 2011;Mani and Bharagava 2016), local sarcomas in various organs (Pohanish 2017), chromosomal abrasions (Bharagava et al. 2018), cystitis, and renal failure (Mani and Bharagava 2016). ...
Article
Full-text available
The projected increase of the global textile industry to USD1002.84 billion in 2027 indicates a simultaneous increase in water pollution due to textile dye-rich voluminous effluents highlighting the requirement of source clean-up. This review analyzes the colossal amount of literature on lab-scale nanoremediation technologies involving iron-based nanoparticles and the mechanistic aspects. However, not many studies are in place with regard to execution because there are several bottlenecks in the scale-up of the technology. This review attempts to identify the limitations of scale-up by focusing on each step of nanoremediation from synthesis of iron-based nanoparticles to their applications. The most prominent appears to be the low economic viability of physico-chemical synthesis of nanoparticles, lack of appropriate toxicity studies of iron-based nanoparticles, and dearth of studies on field applications. It is recommended that above studies should be made not only on lab scale but also on field samples preferably utilizing microbial products based green synthesized iron-based nanoparticles and conducting toxicity studies. Besides, immobilization of the nanoparticles on renewable material greatly enhances the sustainability and economic value of the process. Furthermore, since the chemical composition of dye-rich effluents varies among industries, effluent specific optimization of process parameters and kinetics thereof is also a major prerequisite for scale-up. The value of this review lies in the fact that it brings, for the first time, a comprehensive and critical systematization of various aspects needing attention in order to scale-up such effective nanoremediation processes. Graphical abstract
Article
Water pollution, mainly occurring from manufacturing industries, has been a serious environmental issue in the contemporary world. Dyeing processes produce a massive amount of dye-contaminated effluent; it is a major culprit for water and soil pollution in developing and underdeveloped countries. The azo dye released from industries poses a severe environmental menace by contaminating the aquatic system and adversely impacting human health. Azo dyes are synthetic macromolecules, which are persistent, recalcitrant, and non-biodegradable. Several physicochemical technologies have been proposed for azo dye mitigation. Still, some limitations are observed, such as high operational cost and energy requirement, complicated procedures, incomplete mineralization, and secondary waste generation. Alternately, remediation by microbes is considered a clean, effective, and safe technology to detoxify azo dyes from wastewater. The biological treatment can exploit fungi, yeast, bacteria, and algae, which have received attentiveness because of their eco-friendliness. This review highlights the decolorization and degradation of dyes by various microbes, acting as biological tools against azo dyes. It also discusses the azo dye degradation mechanism and factors affecting the degradations.
Article
The vibronic absorption spectrum of Azure A cation (AA+) in an aqueous solution was calculated using the time‐dependent density functional theory (TD‐DFT). The calculations were performed using all hybrid functionals supported by Gaussian16 software, 6‐31++G(d,p) basis set and polarizable continuum model with external integration formalism (IEFPCM) and solvation model based on solute electron density (SMD) solvent models. The IEFPCM gave significantly underestimated values of λmax in comparison with the experiment, what is a manifestation of the well‐known TD‐DFT “cyanine failure.” However, the SMD made it possible to obtain good agreement between the calculation results and experimental data. The best fit was achieved using the X3LYP functional. The dipole moments and atomic charges of the ground and excited states of the AA+ molecule were calculated. Photoexcitation leads to an increase in the dipole moment of the dye molecule. An insignificant photoinduced electron transfer was found in the central ring of the chromophore of the dye molecule. The vibronic absorption spectrum of Azure A cation in an aqueous solution was calculated using the TD‐DFT. All hybrid functionals with IEFPCM solvent model gave significantly underestimated theoretical λmax values. X3LYP functional with SMD solvent model and 6‐31++G(d,p) gave good agreement of theoretical λmax value with experimental one. Photoexcitation leads to an increase in the dipole moment of the dye. An insignificant photoinduced electron transfer was found in the central ring of the chromophore.
Article
The development of a novel textile sludge based activated carbon (TSBAC) adsorbent and its performance for the treatment of textile dyeing effluent, have been explained in this paper. TSBAC was prepared by the thermal treatment of textile effluent treatment sludge followed by the chemical activation using phosphoric acid. Characterization of TSBAC resulted in enhanced specific surface area (123.65 m²/g) along with the presence of active surface functional groups including −OH, −COOH, −CO. TSBAC showed superior adsorption capacity for methylene blue (123.6 mg/g), reactive red 198 (101.4 mg/g), and reactive yellow 145 (96.8 mg/g) individually, and from the synthetic textile effluent (106 mg/g). The pseudo-second order model and Langmuir isotherm model were found to be fitted well with batch experimental data. The results of the continuous column studies showed that adsorption capacity for methylene blue, reactive red 198, reactive yellow 145 are 101.8 mg/g, 76.6 mg/g, and 75.1 mg/g respectively, and the synthetic textile effluent resulted in an adsorption capacity value of 79.1 mg/g. The reuse potential of TSBAC was proved by effective dye removal up to six reuse cycles. The leachability studies proved that the used adsorbent could be safely disposed of without any harmful effect to the environment.
Chapter
Textile industries are one of the largest income and employment generating industries essentially involved in the production and creation of yarn, cloth, and fabrics. Among the different processes of textile production, finishing process involve the use of synthetic dyes in large concentrations. Unfortunately, inadequacy of the dyeing processes results in the discharge of most of the dyes into natural environment. Due to the complex aromatic structure, these dyes do not degrade within time; persist in the environment and causes harmful effects like soil infertility, contamination of ground water and reduction of light penetration in the water bodies affecting the aquatic ecosystem. In humans, dyes are mutagenic and known to cause cancers, asthma, skin diseases, and neurotoxic effects through bioaccumulation. Therefore, treatment of dyes containing textile effluent is a very important step towards environment and health protection. Among the several treatment strategies, biological methods using plants either alone or in consortium with microorganisms has proven more efficient and cost effective. Phytoremediation of synthetic dyes and other toxic elements using constructed wetland system (CW) is being actively studied and implemented. With this background, present chapter discusses types of dyes, their toxicity, different categories of wetland systems and the current status of constructed wetlands for the management of hazardous textile wastewater.
Article
Full-text available
The spectral study of binding of oxazine (NB) and thiazine (AA, AB, AC, MB and TB O) dyes with cationic cetyltrimethylammonium bromide (CTAB) and non ionic tween 80, surfactant has been done in alkaline medium at lab temperature. All dyes show blue shifting in their λmax value in presence of both CTAB and tween 80. Since, the dye which shows less blue shifting in its λmax value with CTAB/tween 80 is considered more stable. Therefore, the dyes MB, TB O and AA (less blue shifting) are more stable than AB, AC and NB (more blue shifting) in presence of CTAB/tween 80. But, the magnitude of difference between shifted wavelength and its λmax value of all dye are higher with tween 80 in comparison to CTAB. It means the dyes may be more stable with CTAB than tween 80 and the stability of dye-CTAB/tween 80 is directly proportional to the electrical output in photogalvanic cells. The order of stability of these dyes on the basis of blue shifting with CTAB/tween 80 is: MB > AA > TB O > AB> AC> NB. Hence, this type of study may be helpful for choosing a stable dye-surfactant combination and to understand the reason for stability and higher electrical output of the photogalvanic cell for solar energy conversion and storage.
Article
Full-text available
The photochemistry of dye is playing a significant role for understanding the mechanism of electron transfer reactions in photoelectrochemical devices such as photogalvanic cells, DSSC, semiconductor photo-catalysis, photoconductors, etc. Oxazines (Brilliant Cresyl Blue and Nile Blue O) and thiazines (Azur A, Azur B, Azur C, Methylene Blue and Toluidine Blue O) dyes have been used widely as a photosensitizer with and without surfactants in the photogalvanic cells for solar power conversion and storage. Since, the stability and solubility of photosensitizers (dyes) are increased in the presence of surfactant and these properties lead to enhance the electrical output of the photogalvanic cells. Therefore, here we have studied the extent of interaction of different dyes with sodium dodecyl sulphate (SDS), find out the order of stability of dye–SDS on the basis of magnitudes of shifting in λmax of dye monomer and try to correlate order of dye–SDS interaction with already reported electrical output data of photogalvanic cells. Brilliant Cresyl Blue, Nile Blue O, Azur A and TB O have shown red shifting while Azur B, Azur C and MB have shown blue shifting in their λmax value with SDS, which indicates formation of dye–surfactant complex. But, the extent of formation of complex for different dyes with SDS was different due to change in their alkyl groups. Dyes with red shifting have greater stability in excited state as well as higher electrical output data of the cell than dye with blue shifting. On the basis of both red and blue shifting, order of stability of dyes–SDS complex was found as: Brilliant Cresyl Blue > Toluidine Blue O > Azur A > Nile Blue > Azur B > Methylene Blue > Azur C. The order of electrical output values of these dyes in photogalvanic cells have also been supported by literature data in the presence of SDS. Hence, the dye–surfactant complex which would have greater stability in excited state might be more useful for improvement of conversion efficiency and storage capacity of photogalvanic cells in the future.
Chapter
A major component of wastewater generated from various industries is dye. Synthetic dyes are widely used nowadays. Most of these dyes are toxic to environment. They not only cause the death of aquatic flora and fauna, but are also carcinogenic to humans. Stringent colored wastewater can block the passage of sunlight causing harm to aquatic environment. The dyes cause allergies and skin irritations to humans when they come in contact with the dye effluents. Dye is toxic because its chemical structure consists of different aromatic and azo groups. Industries such as textile, rubber, pharmaceutical, paper printing, food, leather, and cosmetics are the major source of generation of colored effluents. Chemical or physical process of dye degradation is not always eco-friendly. Therefore, biological methods are the alternative means to degrade dye. The ability of microorganisms, e.g., bacteria and fungi, to degrade dye has become a new field of interest for researchers because it is relatively cheap and environment friendly method to remove dye from wastewater. There is evidence that many bacteria (for example, Bacillus sp.) and few fungi (for example, Aspergillus sp.) can remove dye from effluents. One of the key enzymes used by microorganisms in this process is oxidase. Due to their enzymatic action, they are capable to degrade dye efficiently. In this book chapter, the microbial degradation of dye-containing wastewater has been studied.
Article
The vibronic absorption spectrum of Azure B (AB) in an aqueous solution is calculated using the time-dependent density functional theory (TD-DFT). The results of calculations are analyzed using all hybrid functionals supported by Gaussian16, the 6-31++G(d,p) basis set, and the IEFPCM and SMD solvent models. The solvent model IEFPCM gave significantly underestimated values of λmax in comparison with the experiment. This is a manifestation of the TD-DFT "cyanine failure". However, the SMD model made it possible to obtain good agreement between the calculation results and experimental data. The best fit was achieved using the X3LYP functional. According to our calculations, the shoulder in the visible absorption spectrum of AB has a vibronic origin. However, the calculated shoulder is weaker than the experimental one. Explicit assignment of two water molecules, which form strong hydrogen bonds with a dye molecule, leads to a shift of the calculated absorption spectrum to longer wavelengths by approximately 17 nm but does not lead to an improvement in its shape. Comparative analysis of the calculated vibronic absorption spectra of Azure B with those obtained earlier for Azure A and methylene blue showed that the presence and intensity of the short-wavelength shoulder are determined by the location of the bands of higher vibronic transitions relative to the band of the 00→0⁰ main transitions. Photoexcitation leads to an increase in the dipole moment of the dye molecule. An insignificant photoinduced electron transfer was found in the central ring of the chromophore of the dye molecule.
Article
Full-text available
Dysregulation of cellular transcription and translation is a fundamental hallmark of cancer. As CDK9 and Mnks play pivotal roles in the regulation of RNA transcription and protein synthesis, respectively, they are important targets for drug development. We herein report the cellular mechanism of a novel CDK9 inhibitor CDKI-73 in an ovarian cancer cell line (A2780). We also used shRNA-mediated CDK9 knockdown to investigate the importance of CDK9 in the maintenance of A2780 cells. This study revealed that CDKI-73 rapidly inhibited cellular CDK9 kinase activity and down-regulated the RNAPII phosphorylation. This subsequently caused a decrease in the eIF4E phosphorylation by blocking Mnk1 kinase activity. Consistently, CDK9 shRNA was also found to down-regulate the Mnk1 expression. Both CDKI-73 and CDK9 shRNA decreased anti-apoptotic proteins Mcl-1 and Bcl-2 and induced apoptosis. The study confirmed that CDK9 is required for cell survival and that ovarian cancer may be susceptible to CDK9 inhibition strategy. The data also implied a role of CDK9 in eIF4E-mediated translational control, suggesting that CDK9 may have important implication in the Mnk-eIF4E axis, the key determinants of PI3K/Akt/mTOR- and Ras/Raf/MAPK-mediated tumorigenic activity. As such, CDK9 inhibitor drug candidate CDKI-73 should have a major impact on these pathways in human cancers.
Article
Full-text available
MicroRNAs (miRNAs) are small 22-25 nucleotides long non-coding RNAs, that are conserved during evolution, and control gene expression in metazoan animals, plants, viruses, and bacteria primarily at post-transcriptional and transcriptional levels. MiRNAs ultimately regulate target gene expression by degrading the corresponding mRNA and/or inhibiting their translation. Currently, the critical functions of miRNAs have been established in regulating immune system, cell proliferation, differentiation and development, cancer and cell cycle by as yet unknown control mechanism. MiRNAs play an essential role in malignancy, and as tumour suppressors and oncogenes. Thus, discovery of new miRNAs will probably change the landscape of cancer genetics. Significantly different miRNA profiles can be assigned to various types of tumours, which could serve as phenotypic signatures for different cancers for their exploitation in cancer diagnostics, prognostics and therapeutics. If miRNA profiles can accurately predict malignancies, this technology could be exploited as a tool to surmount the diagnostic challenges. This review provides comprehensive and systematic information on miRNA biogenesis and their implications in human health.
Article
Full-text available
The plant alkaloid aristololactam-β-d-glucoside and the anticancer chemotherapy drug daunomycin are two sugar bearing DNA binding antibiotics. The binding of these molecules to three double stranded ribonucleic acids, poly(A)·poly(U), poly(I)·poly(C) and poly(C)·poly(G), was studied using various biophysical techniques. Absorbance and fluorescence studies revealed that these molecules bound non-cooperatively to these ds RNAs with the binding affinities of the order 10(6) for daunomycin and 10(5) M(-1) for aristololactam-β-d-glucoside. Fluorescence quenching and viscosity studies gave evidence for intercalative binding. The binding enhanced the melting temperature of poly(A)·poly(U) and poly(I)·poly(C) and the binding affinity values evaluated from the melting data were in agreement with that obtained from other techniques. Circular dichroism results suggested minor conformational perturbations of the RNA structures. The binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived from calorimetry were in agreement with that obtained from spectroscopic data. Daunomycin bound all the three RNAs stronger than aristololactam-β-d-glucoside and the binding affinity varied as poly(A)·poly(U) > poly(I)·poly(C) > poly(C)·poly(G). The temperature dependence of the enthalpy changes yielded negative values of heat capacity changes for the complexation suggesting substantial hydrophobic contribution to the binding process. Furthermore, an enthalpy-entropy compensation behavior was also seen in all systems. These results provide new insights into binding of these small molecule drugs to double stranded RNA sequences.
Article
Full-text available
The mode, mechanism and energetics of interaction of phenosafranine, the planar, cationic and rigid phenazium dye to calf thymus DNA was investigated from absorption, fluorescence, circular dichroism, isothermal titration calorimetry, thermal melting, and viscosity. The study revealed non-cooperative binding of the dye to DNA with an affinity in the range (3.81–4.22) × 105 M−1 as observed from diverse techniques and obeying neighbor exclusion principle. The stoichiometry of binding was characterized to be one phenosafranine molecule per two base pairs. The binding was characterized by strong stabilization of DNA against thermal strand separation, large intrinsic circular dichroic changes of DNA by itself and the generation of induced circular dichroism for the optically inactive phenosafranine molecules. Hydrodynamic and fluorescence quenching studies revealed strong evidence that the phenosafranine molecules are intercalated between every alternate base pairs of calf thymus DNA. Isothermal titration calorimetry studies suggested that the binding was exothermic and favoured by both negative enthalpy and positive entropy changes. This study for the first time presents the complete molecular aspects and energetics of phenosafranine complexation to DNA as model for intercalative drug–DNA interaction.
Article
Full-text available
Emerging evidence suggests an important role for human epidermal keratinocytes in innate immune mechanisms against bacterial and viral skin infections. The proinflammatory effect of viral infections can be mimicked by double-stranded RNA (dsRNA). Herein, we demonstrate that keratinocytes express all known dsRNA sensing receptors at a constitutive and inducible level, and that they use several downstream signaling pathways leading to a broad pattern of gene expression, not only proinflammatory and immune response genes under the control of NF-kappaB, but also genes under transcriptional control of IRF3. As a consequence, dsRNA, a stimulus for TLR3, protein kinase R (PKR), and the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5, induces a status of antiviral defense in keratinocytes. Using inhibitors for the various dsRNA signaling pathways and specific small interfering RNA for TLR3, RIG-I, and MDA5, we demonstrated that in human keratinocytes, TLR3 seems to be necessary for NF-kappaB but not for IRF3 activation, whereas RIG-I and MDA5 are crucial for IRF3 activation. PKR is essential for the dsRNA response in both signaling pathways and thus represents the central antiviral receptor for dsRNA stimulation. Moreover, human keratinocytes up-regulate TLR7, the receptor for single-stranded RNA, in response to stimulation with dsRNA, which renders keratinocytes functionally responsive to the TLR7 agonist gardiquimod, a member of the imidazoquinoline antiviral immune response modifier family. Thus, in addition to building a physical barrier against infectious pathogens, keratinocytes are specially equipped with a full antiviral defense program that enables them to efficiently target viral infections of the skin.
Article
Full-text available
The biological stains, methylene blue and its metabolite azure B, were evaluated as anti-tumor and anti-inflammatory agents. Azur B, administered in drinking water to tumor-bearing mice, inhibited the growth of transplanted tumors and the growth of primary tumors induced by methylcholanthrene. Inhibition of growth of primary tumors was observed only in female mice. Azure B also reduced the wet weight of carrageenin-induced granulomas in rats. Azure B, given intravenously to BCG-sensitized mice 15 minutes prior to challenge with lipopolysaccharide, decreased TNF production (to 10% of control values) and prevented death from endotoxic shock. Methylene blue decreased TNF production (to 50% of control values) but did not protect the animals from endotoxic shock. Our results suggest that some of the effects previously ascribed to methylene blue are probably mediated via its metabolite, i.e. azure B. Low toxicity and easy administration of the dyes explain their use in clinical settings.
Article
Full-text available
In 1891 Guttmann and Ehrlich (P. Guttmann and P. Ehrlich, Berlin Klin. Wochenschr. 28:953-956, 1891) were the first to report the antimalarial properties of a synthetic, rather than a natural, material when they described the clinical cure of two patients after oral administration of a thiazine dye, methylene blue. Since that time, sporadic reports of the antimalarial properties of several xanthene and azine dyes related to methylene blue have been noted. We report here the results from a reexamination of the antimalarial properties of methylene blue. Janus green B, and three rhodamine dyes and disclose new antimalarial data for 16 commercially available structural analogs of these dyes. The 50% inhibitory concentrations for the chloroquine-susceptible D6 clone and SN isolate and the chloroquine-resistant W2 clone of Plasmodium falciparum were determined by the recently described parasite lactate dehydrogenase enzyme assay. No cross-resistance to chloroquine was observed for any of the dyes. For the 21 dyes tested, no correlation was observed between antimalarial activity and cytotoxicity against KB cells. No correlation between log P (where P is the octanol/water partition coefficient) or relative catalyst efficiency for glucose oxidation and antimalarial activity or cytotoxicity was observed for the dyes as a whole or for the thiazine dyes. The thiazine dyes were the most uniformly potent structural class tested, and among the dyes in this class, methylene blue was notable for both its high antimalarial potency and selectivity.
Article
Full-text available
Translational control is a prevalent means of gene regulation during Drosophila oogenesis and embryogenesis. Multiple maternal mRNAs are localized within the oocyte, and this localization is often coupled to their translational regulation. Subsequently, translational control allows maternally deposited mRNAs to direct the early stages of embryonic development. In this review we outline some general mechanisms of translational regulation and mRNA localization that have been uncovered in various model systems. Then we focus on the posttranscriptional regulation of four maternal transcripts in Drosophila that are localized during oogenesis and are critical for embryonic patterning: bicoid (bcd), nanos (nos), oskar (osk), and gurken (grk). Cis- and trans-acting factors required for the localization and translational control of these mRNAs are discussed along with potential mechanisms for their regulation.
Article
Full-text available
miRNAs (microRNAs) were first discovered as critical regulators of developmental timing events in Caenorhabditis elegans. Subsequent studies have shown that miRNAs and cellular factors necessary for miRNA biogenesis are conserved in many organisms, suggesting the importance of miRNAs during developmental processes. Indeed, mutations in the miRNA-processing pathway induce pleiotropic defects in development, which accompany perturbation of correct expression of target genes. However, control of gene expression in development is not the only function of miRNAs. Recent work has provided new insights into the role of miRNAs in various biological events, including aging and cancer. C. elegans continues to be helpful in facilitating a further understanding of miRNA function in human diseases.
Article
Full-text available
The modulation of gene expression by small non-coding RNAs is a recently discovered level of gene regulation in animals and plants. In particular, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) have been implicated in various aspects of animal development, such as neuronal, muscle and germline development. During the past year, an improved understanding of the biological functions of small non-coding RNAs has been fostered by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators of animal development and are potential human disease loci.
Article
Full-text available
Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals.
Article
A spectrophotometric method for ascertaining the presence of anionic detergent in milk using azure A dye is described. The method requires brief mixing of milk, azure A dye and EDTA solutions, followed by addition of chloroform and subsequent centrifugation. The blue colour in the chloroform phase was measured at 635 nm. The limits of detection (LOD) and limit of quantification (LOQ) for sodium dodecylbenzene sulfonate in milk were 10 and 20 mg L−1, respectively and for a laboratory grade anionic detergent, labolene, 50 and 100 mg L−1, respectively. Recovery of detergent in spiked milk samples was quantitative. The presence of other additives, i.e., NaOH, NaHCO3, KNO3 and NaCl at levels of 250, 750, 500 and 500 mg L−1 milk, respectively, did not interfere with estimation of detergent. The method is also applicable to milk samples stored in the presence of formalin and sodium azide for 39 days at 4 °C.
Article
Mitomycin C (MC), mitomycin A, porfiromycin, BMY-25067, and BMY-25287, antitumor antibiotics collectively termed ''mitosanes'', were found to have no appreciable binding affinity to various natural and synthetic DNAs, as tested by UV spectrophotometry and equilibrium dialysis. Further tests of DNA binding applied to MC including thermal melting measurements, displacement of ethidium fluorescence, and unwinding of closed circular DNA were similarly negative. In contrast, 2,7-diaminomitosene (2,7-DAM), a major end product of the reductive activation of MC, binds to the same series of DNAs by all of these criteria. In the presence of DNA its UV absorbance at the 313 nm maximum decreased and underwent a slight red shift. This effect was used for determining DNA binding constants (K-b) by the spectrophotometric titration method. At pH 6.0 the K(b)s of three natural DNAs with varying GC content, as well as poly(dA-dT). poly(dA-dT), and poly(dG-dC). poly(dG-dC), were all in the range of (1.2-5.3) x 10(4) (M nucleotide)(-1), with no apparent specificity of binding. Poly(dG-m5dC). poly(dG-m5dC) displayed a slightly higher K-b ((7.5-8.4) x 10(4)). Binding of other, closely related mitosenes was tested to calf thymus DNA by equilibrium dialysis. Neither the presence of a 1-OH substituent, removal of the 10-carbamoyl group, nor methylation of the 2-amino group modifies the binding affinity of the mitosenes significantly. The 1-phosphate substituent abolishes binding. The binding of 2,7-DAM to DNA increased with decreasing pH and decreasing ionic strength. It was determined that 2,7-DAM is protonated at the 2-amino group with a pK(a) = 7.55, and this correlated well with the observed pH dependence of the binding, indicating that the binding affinity has a strong electrostatic component. This was confirmed by the finding that the extrapolated K-b to 1 M Na+ concentration diminishes to only 10% of the value of K-b at 0.01 M Na+ concentration. Viscosity tests showed conclusively that 2,7-DAM intercalates in DNA, in a nonspecific manner. DNA binding by 2,7-DAM is shown to be a close model of the binding of the reduced activated form of MC, previously characterized indirectly [Teng, S. P., Woodson, S. A., and Crothers, D. M. (1989) Biochemistry 28, 3901-3907]. The nonspecific precovalent binding of the active form may serve in the cell to concentrate the drug at its critical target, DNA. A CpG-specific minor groove binding mode, previously suggested to explain the observed CpG specificity of the covalent alkylation [Kumar, S., Lipman, R., and Tomasz, M. (1992) Biochemistry 31, 1399-1407], is presumably masked by the stronger, bulk nonspecific binding described here.
Article
Association constants for inclusion complexes are commonly determined by varying the analytical concentration of the “host” species while maintaining the analytical concentration of “guest” species constant. In implementing the experiment, commonly referred to as Benesi−Hildebrand analysis, conditions are controlled such that all necessary concentration requirements are fulfilled and the guest molecule in the unbound state does not contribute appreciably to the overall signal (<5%). In this paper, we demonstrate that this commonly accepted constraint, while clearly important, is insufficient for the accurate determination of association constants. Systematic error introduced by variation in the host species concentration, and therefore the equilibrium concentration of unbound guest molecules, results in overestimation of the complexation constant. These errors in the association constant determination appear to be the most troublesome when steady-state fluorescence or absorbance measurements, two of the most common techniques, are utilized. We demonstrate that correction of this systematic error can be accomplished by iterative correction for the equilibrium concentration of the unbound guest.
Article
The interaction between double-stranded (ds) T2 DNA and the fluorescent dyes YO (oxazole yellow) and YOYO (dimer of oxazole yellow) has been studied with optical spectroscopic methods. Flow linear dichroism (LD) spectra of YO-DNA and YOYO-DNA complexes show that, at mixing ratios dye:DNA base below 0.20 and 0.125 for YO-DNA and YOYO-DNA, respectively, the long axis in the YO chromophore is approximately perpendicular to the DNA helix axis, an orientation consistent with intercalation. This conclusion is supported by the induced negative circular dichroism (CD), the transfer of energy from the DNA bases to the bound YOYO, and the unwinding of supercoiled DNA by YOYO. At higher mixing ratios, a CD exciton appears and the steady increase in the reduced linear dichroism amplitude during the intercalation phase is changed to a decrease, suggesting that a second binding mode starts to contribute. For YO, the exciton has the expected pattern for dimeric interaction between chromophores bound to the surface of the DNA. For YOYO, the new binding mode appears at a mixing ratio where, if the dye is assumed to follow the nearest neighbor exclusion principle, all intercalation sites are filled up. Thus, this second binding mode is proposed to be an external binding mode. Fluorescence anisotropy measurements show that the limiting anisotropy value (mixing ratio dye:base --> 0) for YO-DNA is about twice that of YOYO-DNA. In addition, the decrease in fluorescence anisotropy with the mixing ratio is much stronger for YO-DNA compared to YOYO-DNA. These observations have been explained in terms of depolarization of the emission due to Forster energy transfer between the intercalated chromophores.
Article
The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNAphe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.
The ORD and CD spectra and fluorescence measurements of complexes between poly(I) and poly(C) at different pH and ionic strength are reported. The evolution of optical activity and fluorescence closely follows the results obtained earlier on this system by spectrophotometric methods. These data clearly distinguish three different complexes: poly(I) · poly(C), poly(I) · poly(C) · poly(C+) and poly(I) · poly(C+). The latter is probably a reverse Hoogsteen paired helix. Comparison with published data on poly(dI) · poly(dC) suggest that the deoxypolymer may be a non-Watson-Crick helix.
Article
Methylene blue (MB) has been shown to act at multiple cellular and molecular targets and as a result possesses diverse medical applications. Among these is a high potency reversible inhibition of monoamine oxidase A (MAO-A) that may, at least in part, underlie its adverse effects but also its psycho- and neuromodulatory actions. MB is metabolized to yield N-demethylated products of which azure B, the monodemethyl species, is the major metabolite. Similar to MB, azure B also displays a variety of biological activities and may therefore contribute to the pharmacological profile of MB. Based on these observations, the present study examines the interactions of azure B with recombinant human MAO-A and -B. The results show that azure B is a potent MAO-A inhibitor (IC₅₀=11 nM), approximately 6-fold more potent than is MB (IC₅₀=70 nM) under identical conditions. Measurements of the time-dependency of inhibition suggest that the interaction of azure B with MAO-A is reversible. Azure B also reversibly inhibits the MAO-B isozyme with an IC₅₀ value of 968 nM. These results suggest that azure B may be a hitherto under recognized contributor to the pharmacology and toxicology of MB by blocking central and peripheral MAO-A activity and as such needs to be considered during its use in humans and animals.
Article
The relevance of the non-coding genome to human disease has mainly been studied in the context of the widespread disruption of microRNA (miRNA) expression and function that is seen in human cancer. However, we are only beginning to understand the nature and extent of the involvement of non-coding RNAs (ncRNAs) in disease. Other ncRNAs, such as PIWI-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), transcribed ultraconserved regions (T-UCRs) and large intergenic non-coding RNAs (lincRNAs) are emerging as key elements of cellular homeostasis. Along with microRNAs, dysregulation of these ncRNAs is being found to have relevance not only to tumorigenesis, but also to neurological, cardiovascular, developmental and other diseases. There is great interest in therapeutic strategies to counteract these perturbations of ncRNAs.
Article
The ability of RNA to both store genetic information and catalyse chemical reactions has led to the hypothesis that it predates DNA and proteins. While there is no doubt that RNA is capable of storing the genetic information of a primitive organism, only two classes of reactions-phosphoryl transfer and peptide bond formation-have been observed to be catalysed by RNA in nature. However, these naturally occurring ribozymes use a wide range of catalytic strategies that could be applied to other reactions. Furthermore, RNA can bind several cofactors that are used by protein enzymes to facilitate a wide variety of chemical processes. Despite its limited functional groups, these observations indicate RNA is a versatile molecule that could, in principle, catalyse the myriad reactions necessary to sustain life.
Article
MicroRNAs (miRNAs) have emerged as critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than a third of all protein coding genes, and they have been implicated in the control of virtually all biological processes, including the biology of stem cells. The essential roles of miRNAs in the control of pluripotent stem cells were clearly established by the finding that embryonic stem (ES) cells lacking proteins required for miRNA biogenesis exhibit defects in proliferation and differentiation. Subsequently, the function of numerous miRNAs has been shown to control the fate of ES cells and to directly influence critical gene regulatory networks controlled by pluripotency factors Sox2, Oct4, and Nanog. Moreover, a growing list of tissue-specific miRNAs, which are silenced or not processed fully in ES cells, has been found to promote differentiation upon their expression and proper processing. The importance of miRNAs for ES cells is further indicated by the exciting discovery that specific miRNA mimics or miRNA inhibitors promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Although some progress has been made during the past two years in our understanding of the contribution of specific miRNAs during reprogramming, further progress is needed since it is highly likely that miRNAs play even wider roles in the generation of iPS cells than currently appreciated. This review examines recent developments related to the roles of miRNAs in the biology of pluripotent stem cells. In addition, we posit that more than a dozen additional miRNAs are excellent candidates for influencing the generation of iPS cells as well as for providing new insights into the process of reprogramming.
Article
Riboswitches are cis-encoded, cis-acting RNA elements that directly sense a physiological signal. Signal response results in a change in RNA structure that impacts gene expression. Elements of this type play an important role in bacteria, where they regulate a variety of fundamental cellular pathways. Riboswitch-mediated gene regulation most commonly occurs by effects on transcription attenuation, to control whether a full-length transcript is synthesized, or on translation initiation, in which case the transcript is constitutively synthesized but binding of the translation initiation complex is modulated. An overview of the role of riboswitch RNAs in bacterial gene expression will be provided, and a few examples are described in more detail to illustrate the types of mechanisms that have been uncovered.
Article
The interaction of two natural protoberberine plant alkaloids berberine and palmatine and a synthetic derivative coralyne to three double stranded ribonucleic acids, poly(A). poly(U), poly(I).poly(C) and poly(C).poly(G) was studied using various biophysical techniques. Absorbance and fluorescence studies showed that the alkaloids bound cooperatively to these RNAs with the binding affinities of the order 10(4) M(-1). Circular dichroic results suggested that the conformation of poly(A). poly(U) was perturbed by all the three alkaloids, that of poly(I).poly(C) by coralyne only and that of poly(C).poly(G) by none. Fluorescence quenching studies gave evidence for partial intercalation of berberine and palmatine and complete intercalation of coralyne to these RNA duplexes. Isothermal titration calorimetric studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived were in agreement with the overall binding affinity from spectral data. The binding of all the three alkaloids considerably stabilized the melting of poly(A). poly(U) and poly(I).poly(C) and the binding data evaluated from the melting data were in agreement with that obtained from other techniques. The overall binding affinity of the alkaloids to these double stranded RNAs varied in the order, berberine = palmatine < coralyne. The temperature dependence of the enthalpy changes afforded large negative values of heat capacity changes for the binding of palmatine and coralyne to poly(A).poly(U) and of coralyne to poly(I).poly(C), suggesting substantial hydrophobic contribution in the binding process. Further, enthalpy-entropy compensation was also seen in almost all the systems that showed binding. These results further advance our understanding on the binding of small molecules that are specific binders to double stranded RNA sequences.
Article
CD spectra and difference-CD spectra of (a) tvo DNA*RNA hybrid duplexes (poly(r(A)·d(U)) and poly(r(A)·d(T)) and (b) three hybrid triplexes (poly-(d(T)·r(A)·d(T)), poly(r(U)·d(A)·r(U)J), and poly(r(T)·d(A)·r(T)J) were obtained and compared with CD spectra of six A·U- and A·T-containing duplex and triplex RNAs and DNAs. We found that the CD spectra of the homopolymer duplexes above 260 nm were correlated with the type of base pair present (A·U or A·T) and could be interpreted as the sum of the CD contributions of the single strands plus a contribution due to base pairing. The spectra of the duplexes below 235 nm were related to the polypurine strands present (poly-(r(A) or poly(d(A)). We interpret the CD intensity in the intermediate 255–235 nm region of these spectra to be mainly due to stacking of the consti-tuent polypurine strands. Three of the five hybrids (poly(r(A)·d(U)), poly-(r(A)·d(T)), and poly(d(T)·r(A)·d(T)) were found to have heteronomous conformations, while poly(r(U)·d(A)·r(U)) was found to be the most A-like and poly(r(T)·d(A)·r(T)), the least A-like.
Article
In Alzheimer disease (AD) the microtubule-associated protein tau is redistributed exponentially into paired helical filaments (PHFs) forming neurofibrillary tangles, which correlate with pyramidal cell destruction and dementia. Amorphous neuronal deposits and PHFs in AD are characterized by aggregation through the repeat domain and C-terminal truncation at Glu-391 by endogenous proteases. We show that a similar proteolytically stable complex can be generated in vitro following the self-aggregation of tau protein through a high-affinity binding site in the repeat domain. Once started, tau capture can be propagated by seeding the further accumulation of truncated tau in the presence of proteases. We have identified a nonneuroleptic phenothiazine previously used in man (methylene blue, MB), which reverses the proteolytic stability of protease-resistant PHFs by blocking the tau-tau binding interaction through the repeat domain. Although MB is inhibitory at a higher concentration than may be achieved clinically, the tau-tau binding assay was used to identify desmethyl derivatives of MB that have Ki values in the nanomolar range. Neuroleptic phenothiazines are inactive. Tau aggregation inhibitors do not affect the tau-tubulin interaction, which also occurs through the repeat domain. Our findings demonstrate that biologically selective pharmaceutical agents could be developed to facilitate the proteolytic degradation of tau aggregates and prevent the further propagation of tau capture in AD.
Article
A plethora of post-transcriptional mechanisms are involved in essential steps in the pathway of genetic information expression in eukaryotes. These processes are specified by cis-acting signals on RNAs and are mediated by specific trans-acting factors, including RNA-binding proteins and small complementary RNAs. Recent information has begun to define the molecular mechanisms by which RNA-binding proteins recognize specific RNA sequences and influence the processing and function of RNA molecules.
Article
Bacterial small, untranslated RNAs are important regulators that often act to transmit environmental signals when cells encounter suboptimal or stressful growth conditions. These RNAs help modulate changes in cellular metabolism to optimize utilization of available nutrients and improve the probability for survival.
Article
The combination in solution of DNA with small amounts of acridine, proflavine, or acridine orange results in markedly enhanced viscosity and a diminution of the sedimentation coefficient of the DNA. These changes are contrary to those expected on the basis of aggregation or simple electrostatic effects. Characteristic changes, which suggest considerable modification of the usual helical structure of DNA, are found in the X-ray diffraction patterns of fibers of the complex with proflavine.It is inferred that these compounds, which are potent mutagens, are intercalated between adjacent nucleotide-pair layers by extension and unwinding of the deoxyribose-phosphate backbone. The hydrodynamic changes are the consequence of the diminished bending between layers, the lengthening of the molecule, and the diminished length-specific mass. The effects are fully reversible at ordinary temperatures. The proposed structural change is compatible with the normal restrictions on bond lengths, angles, and non-bonded contacts, and maintenance of the hydrogen-bonded base pairs perpendicular to the axis of the molecule.Another singly charged dye, pinacyanol, fails to elicit the effects of intercalation, yielding only lowered viscosity and increased sedimentation coefficient.
Article
A new paradigm of RNA-directed gene expression regulation has emerged recently, profound in scope but arresting in the apparent simplicity of its core mechanism. Cells express numerous small ( approximately 22 nucleotide) RNAs that act as specificity determinants to direct destruction or translational repression of their mRNA targets. These small RNAs arise from processing of double-stranded RNA by the Dicer nuclease and incorporate with proteins that belong to the Argonaute family. Small RNAs might also target and silence homologous DNA sequences. The immense potential of small RNAs as controllers of gene networks is just beginning to unfold.
Article
Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of gene silencing that are collectively referred to as RNA silencing or RNA interference. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms. Many components of the RNA silencing machinery still need to be identified and characterized, but a more complete understanding of the process is imminent.
Article
RNA interference is a highly conserved pathway mediating sequence-specific RNA degradation. In plants, the short RNA intermediates of this pathway can also drive transcriptional silencing of target genes by DNA methylation. Until recently, there was no evidence that a similar pathway operated in mammals; two new studies suggest that small RNAs can direct DNA methylation and chromatin modification in human cells. Although further investigation is required to determine how widespread RNA-directed DNA methylation is in mammals, the findings raise the possibility that this pathway, far from being merely a curiosity of plant systems, is a conserved mechanism for control of gene expression.
Article
Protonation-induced conformational changes in natural DNAs of diverse base composition under the influence of low pH, low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH3.40, 10mM [Na+], and at 5 degrees C, all natural DNAs irrespective of base composition adopted an unusual and stable conformation remarkably different from the canonical B-form conformation. This protonated conformation has been characterized to have unique absorption and circular dichroic spectral characteristics and exhibited cooperative thermal melting profiles with decreased thermal melting temperatures compared to their respective B-form counterparts. The nature of this protonated structure was further investigated by monitoring the interaction of the plant alkaloid, berberine that was previously shown from our laboratory to differentially bind to B-form and H(L)-form of poly[d(G-C)] [Bioorg. Med. Chem.2003, 11, 4861]. Binding of berberine to protonated conformation of natural DNAs resulted in intrinsic circular dichroic changes as well as generation of induced circular dichroic bands for the bound berberine molecule with opposite signs and magnitude compared with B-form structures. Nevertheless, the binding of the alkaloid to both the B and protonated forms was non-linear and non-cooperative as revealed from Scatchard plots derived from spectrophotometric titration data. Steady state fluorescence studies on the other hand showed remarkable increase of the rather weak intrinsic fluorescence of berberine on binding to the protonated structure compared to the B-form structure. Taken together, these results suggest that berberine can detect the formation of significant population of H(L)-form structures under the influence of protonation irrespective of heterogeneous base compositions in natural DNAs.
Article
The natural RNA enzymes catalyse phosphate-group transfer and peptide-bond formation. Initially, metal ions were proposed to supply the chemical versatility that nucleotides lack. In the ensuing decades, structural and mechanistic studies have substantially altered this initial viewpoint. Whereas self-splicing ribozymes clearly rely on essential metal-ion cofactors, self-cleaving ribozymes seem to use nucleotide bases for their catalytic chemistry. Despite the overall differences in chemical features, both RNA and protein enzymes use similar catalytic strategies.
Article
A novel organic-inorganic nanocomposite of methylene blue (MB) and silicon oxide was synthesized and characterized by TEM, FTIR, and UV-vis. The as-prepared material was able to transfer the electron of the MB to electrode and was different from other SiO2 spheres structurally. It can be used as mediator to construct a biosensor with horseradish peroxidase (HRP) coimmobilized in the gelatine matrix and cross-linked with formaldehyde. The resulting biosensor exhibited fast amperometric response and good stability to hydrogen peroxide (H2O2). The linear range for H2O2 determination was from 1 x 10(-5) to 1.2 x 10(-3) M, with a detection limit of 4 x 10(-6) M based on S/N = 3. Moreover, the lifetime is more than 3 months under dry conditions at 4 degrees C.
Article
Polymorphic RNA conformations may serve as potential targets for structure specific antiviral agents. As an initial step in the development of such drugs, the interaction of a wide variety of compounds which are characterized to bind to DNA through classical or partial intercalation or by mechanism of groove binding, with the A-form and the protonated form of poly(rC).poly(rG), been evaluated by multifaceted spectroscopic and viscometric techniques. Results of this study suggest that (i) ethidium intercalates to the A-form of RNA, but does not intercalate to the protonated form, (ii) methylene blue intercalates to the protonated form of the RNA but does not intercalate to the A-form, (iii) actinomycin D does not bind to either conformations of the RNA, and (iv) berberine binds to the protonated form by partial intercalation process, while its binding to the A-form is very weak. The DNA groove binder distamycin A has much higher affinity to the protonated form of the RNA compared to the A-form and binds to both structures by non-intercalative mechanism. We conclude that the binding affinity characteristics of these DNA binding molecules to the RNA conformations are vastly different and may serve as data for the development of RNA based antiviral drugs.
Article
MicroRNAs (miRNAs) are a large class of small RNAs that function as negative gene regulators in eukaryotes. They regulate diverse biological processes, and bioinformatics data indicate that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway. In addition to the roles in ontogeny, recent evidence has suggested the possibility that miRNAs have huge impacts on animal phylogeny. The dramatically expanding repertoire of miRNAs and their targets appears to be associated with major body-plan innovations as well as the emergence of phenotypic variation in closely related species. Research in the area of miRNA phylogenetic conservation and diversity suggests that miRNAs play important roles in animal evolution, by driving phenotypic variation during development.
Article
MicroRNAs (miRNAs) are genomically encoded small non-coding RNAs that regulate flow of genetic information by controlling translation or stability of mRNAs. Recent recognition that many miRNAs are expressed in a tissue-specific manner during development of organisms, from worms to humans, has revealed a novel mechanism by which the proteome is regulated during the dynamic events of cell-lineage decisions and morphogenesis. Advances in the understanding of miRNA biogenesis, target recognition and participation in regulatory networks demonstrate a role for miRNAs in lineage decisions of progenitor cells and organogenesis. Future discoveries in this area are likely to reveal developmental-regulation and disease mechanisms related to miRNAs.
Article
The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.
Article
MiRNAs are a conserved class of non-coding RNAs that negatively regulate gene expression post-transcriptionally. Although their biological roles are largely unknown, examples of their importance in cancer, metabolic disease, and viral infection are accumulating, suggesting that they represent a new class of drug targets in these and likely many other therapeutic areas. Antisense oligonucleotide approaches for inhibiting miRNA function and siRNA-like technologies for replacement of miRNAs are currently being explored as tools for uncovering miRNA biology and as potential therapeutic agents. The next few years should see significant progress in our understanding of miRNA biology and the advancement of the technology for therapeutic modulation of miRNA activity.
Article
The heart is among the most conserved organs of the body and is susceptible to defects more than any other organ. Heart malformations, in fact, occur in roughly 1% of newborns. Moreover, cardiovascular disease arising during adult life is among the main causes of morbidity and mortality in developed countries. It is not surprising, therefore, that much effort is being channeled into understanding the development, physiology, and pathology of the cardiovascular system. MicroRNAs, a newly discovered class of small ribonucleotide-based regulators of gene expression, are being implicated in an increasing number of biological processes, and the study of their role in cardiovascular biology is just beginning. Here, we briefly overview microRNAs in general and report on the recent findings regarding their importance for the heart and vasculature, in particular. The new insights that are being gained will permit not only a greater understanding of cardiovascular pathologies but also, hopefully, the development of novel therapeutic strategies.