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Owing to the diverse biological application, photophysical behaviour of Hoechst 33258 (H33258) is an important topic in biophysical research. Here, we have reported the photophysical behavior of H33258 in model biomembrane mimicking micellar microenvironment. In order to understand the molecular mechanism of the interaction of H33258 with micelles, we have used cationic (Hexadecetyltrimethyl ammonium bromide (CTAB)), anionic (sodium dodecyl sulphate (SDS)), and non‐ionic (t‐octylphenoxypolyoxyethanol, Triton‐X100 (TX 100)) micelles. From the steady‐state emission study the evaluated apparent binding constant (Kb) values for the complex formation between H33258 and CTAB, SDS and TX 100 micelle were (1.45±0.42)×105, (31.95±4.89)×105 and (3.10±0.73)×105 M−1, respectively. Thus, it was found that the Kb value was greater in the case of anionic micelle than non‐ionic and cationic micelles. Such differences in Kb values were due to the electrostatic interaction and hydrogen bond formation between the probe molecule and micelles. In addition, time‐resolved fluorescence decay and time‐resolved fluorescence anisotropy studies were performed to understand the dynamical behavior of H33258 in presence of micelles. The interaction of H33258 with micelles might be useful to study the structure and dynamics of different bio‐membranes and to formulate new drug delivery system. [ABSTRACT FROM AUTHOR]

Using a computer modeling approach, we proposed a structure for a potential GC-specific DNA ligand, which could form a complex with DNA in the minor groove similar to that formed by Hoechst 33258 at DNA AT-enriched sites. According to this model, MBoz2A, a bisbenzoxazole ligand, was synthesized. The results of spectrophotometric methods supported the complex formation of the compound under study with DNA differing in the nucleotide composition. [ABSTRACT FROM AUTHOR]

A fundamental possibility of using the ability of low-molecular-weight DNA molecules to form optically active cholesteric liquid-crystalline dispersions (CLCDs) to estimate the efficacy of DNA-binding radioprotectors is studied. On the example of the well-known dye Hoechst 33 258, which interacts with DNA by the minor-groove-binding and has pronounced radioprotective properties, it is shown that the change in the amplitude of anomalous signal of circular dichroism of the CLCDs formed from DNA molecules preliminarily irradiated with X-rays in the absence and in the presence of the test compound makes it possible to estimate the value of its protective effect. [ABSTRACT FROM AUTHOR]

The design and production of biodegradable and sustainable non-toxic materials for solar-energy harvesting and conversion is a significant challenge. Here, our goal was to report the preparation of novel protein/lipid hydrogels and demonstrate their utility in two orthogonal fundamental studies—light harvesting and white-light emission. Our hydrogels contained up to 90% water, while also being self-standing and injectable with a syringe. In one application, we loaded these hydrogels with suitable organic donor-acceptor dyes and demonstrated the energy-transfer cascade among four different dyes, with the most red-emitting dye as the energy destination. We hypothesized that the dyes were embedded in the protein/lipid phase away from the water pools as monomeric entities and that the excitation of any of the four dyes resulted in intense emission from the lowest-energy acceptor. In contrast to the energy-transfer cascade, we demonstrate the use of these gels to form a white-light-emitting hydrogel dye assembly, in which excitation migration is severely constrained. By restricting the dye-to-dye energy transfer, the blue, green, and red dyes emit at their respective wavelengths, thereby producing the composite white-light emission. The CIE color coordinates of the emission were 0.336 and 0.339—nearly pure white-light emission. Thus, two related studies with opposite requirements could be accommodated in the same hydrogel, which was made from edible ingredients by a simple method. These gels are biodegradable when released into the environment, sustainable, and may be of interest for energy applications. [ABSTRACT FROM AUTHOR]

Optically active liquid–crystalline dispersions (LCD) of nucleic acids, obtained by polymer- and salt-induced (psi-) condensation, e.g., by mixing of aqueous saline solutions of low molecular weight DNA (≤106 Da) and polyethylene glycol (PEG), possess an outstanding circular dichroism (CD) signal (so-called psi-CD) and are of interest for sensor applications. Typically, such CD signals are observed in PEG content from ≈12.5% to ≈22%. However, in the literature, there are very conflicting data on the existence of psi-CD in DNA LCDs at a higher content of crowding polymer up to 30–40%. In the present work, we demonstrate that, in the range of PEG content in the system above ≈24%, optically polymorphic LCDs can be formed, characterized by both negative and positive psi-CD signals, as well as by ones rather slightly differing from the spectrum of isotropic DNA solution. Such a change in the CD signal is determined by the concentration of the stock solution of PEG used for the preparation of LCDs. We assume that various saturation of polymer chains with water molecules may affect the amount of active water, which in turn leads to a change in the hydration of DNA molecules and their transition from B-form to Z-form. [ABSTRACT FROM AUTHOR]

Bisbenzimidazoles have a broad spectrum of potential applications: radioprotectors, drug delivery vectors, antiviral agents, etc. At the same time, they seem to be promising fluorescent probes for radiation measurements. Therefore, in the present work, a fluorescent response to X-ray irradiation of Hoechst 33258, one of the most widely known representatives of the bisbenzimidazole family, was studied for the first time. Irradiation of the dye was performed in aqueous and organic solutions (DMSO and glycerol), as well as in their mixtures. It is shown that the reaction of the dye to radiation exposure is very versatile and may be controlled by the solvent properties, which makes it possible to build relationships between the absorbed dose and a wide variety of parameters of its fluorescence signal. For example, irradiation may induce fluorescence quenching caused by the degradation of the dye, a change in the position of the fluorescence band maximum due to the modification of the dye molecules or to the radiation-induced changes in the properties of the medium, as well as a fluorescence flare-up mediated by the changes in pH. [ABSTRACT FROM AUTHOR]

The design and production of biodegradable and sustainable non-toxic materials for solar-energy harvesting and conversion is a significant challenge. Here, our goal was to report the preparation of novel protein/lipid hydrogels and demonstrate their utility in two orthogonal fundamental studies—light harvesting and white-light emission. Our hydrogels contained up to 90% water, while also being self-standing and injectable with a syringe. In one application, we loaded these hydrogels with suitable organic donor-acceptor dyes and demonstrated the energy-transfer cascade among four different dyes, with the most red-emitting dye as the energy destination. We hypothesized that the dyes were embedded in the protein/lipid phase away from the water pools as monomeric entities and that the excitation of any of the four dyes resulted in intense emission from the lowest-energy acceptor. In contrast to the energy-transfer cascade, we demonstrate the use of these gels to form a white-light-emitting hydrogel dye assembly, in which excitation migration is severely constrained. By restricting the dye-to-dye energy transfer, the blue, green, and red dyes emit at their respective wavelengths, thereby producing the composite white-light emission. The CIE color coordinates of the emission were 0.336 and 0.339—nearly pure white-light emission. Thus, two related studies with opposite requirements could be accommodated in the same hydrogel, which was made from edible ingredients by a simple method. These gels are biodegradable when released into the environment, sustainable, and may be of interest for energy applications.

Fluorescence spectroscopy was used to study the interactions of the bisbenzimidazole compound Hoechst 33258 with bovine serum albumin and calf thymus DNA. The fluorescence spectra of these complexes are presented. The maxima in these spectra were used to construct curves for the dependence of the fluorescence intensities of this ligand bound to the indicated protein and DNA on the macromolecule/ligand ratio r. In the case of albumin, the intensity decreases with increasing protein concentration in the low protein concentration region and then increases with increasing protein concentration in the region of higher protein concentrations. In the case of DNA, the intensity steadily increases with increasing DNA concentration. This behavior is attributed to binding of the ligand to both DNA and albumin. [ABSTRACT FROM AUTHOR]

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Bisbenzimidazoles have a broad spectrum of potential applications: radioprotectors, drug delivery vectors, antiviral agents, etc. At the same time, they seem to be promising fluorescent probes for radiation measurements. Therefore, in the present work, a fluorescent response to X-ray irradiation of Hoechst 33258, one of the most widely known representatives of the bisbenzimidazole family, was studied for the first time. Irradiation of the dye was performed in aqueous and organic solutions (DMSO and glycerol), as well as in their mixtures. It is shown that the reaction of the dye to radiation exposure is very versatile and may be controlled by the solvent properties, which makes it possible to build relationships between the absorbed dose and a wide variety of parameters of its fluorescence signal. For example, irradiation may induce fluorescence quenching caused by the degradation of the dye, a change in the position of the fluorescence band maximum due to the modification of the dye molecules or to the radiation-induced changes in the properties of the medium, as well as a fluorescence flare-up mediated by the changes in pH.

Interactions of Hoechst 33258 (H33258) with two synthetic DNA polynucleotides, poly dA–poly dT and poly dG–poly dC, as well as natural calf thymus (ct) DNA were studied using surface‐enhanced Raman spectroscopy (SERS). Given variety of stoichiometry dependent interactions, complexes were prepared in [H33258]/[DNA] molar ratios of 1/1, 1/5, and 1/10 in the silver colloidal suspension. Prior to analysis of binding modes with DNA polynucleotides, aggregation tendency of the dye in the silver colloid was studied in the concentration range 1 × 10−6–1 × 10−4 mol/L. The SERS spectra distinctive of aggregated and monomeric H33258 species were obtained, additionally indicating the form of the molecules bound to DNA. The obtained SERS spectra of H33258 with homo‐polymeric polynucleotides implied a deep insertion of the molecules into the AT minor groove, and a partial intercalation between the GC base pairs achieved by the access through the GC major groove. Spectral characteristics of the dye dominated in the spectra of the complexes with ct‐DNA, most likely due to random AT and GC sequences of the long natural nucleic acid, affecting uniform binding of the dye and consequently the adsorption of the H33258/DNA complex onto the enhancing metallic surface. [ABSTRACT FROM AUTHOR]

Background: Cancer is the second cause of death in developed countries. Colon and breast cancers are among the most prevalent ones. Research focusing on finding new natural products with fewer side effects to fight cancer is increasing. Objectives: The present study aimed to evaluate the cytotoxicity of Centaurea albonitens Turrill methanol extract and its fractions against colon and breast cancer cell lines and a normal cell line of bovine kidney cells. Methods: The methanol extract and petroleum ether, chloroform and aqueous fractions were provided from the aerial parts of C. albonitens by maceration method in three days. Each day the solvent was refreshed. Colon (HT-29) and breast (MCF-7) cell lines were treated with the extract/fractions for 48 h for evaluating the cytotoxic activity by MTT assay. The apoptotic induction potential was also evaluated with the Hoechst 33258 staining method. Results: The most considerable effect was reported from the chloroform fraction with IC50 values of 25.6 and 25.1 μg/mL in MCF-7 and HT-29 cells, respectively. In Hoechst staining, condensed chromatin of the apoptotic cells was observed in both cell lines. Conclusion: Centaurea albonites can be suggested for further cancer research studies.

A comparative investigation of the reaction of the bisbenzimidazole compound Hoechst 33258 and the thiazine dye methylene blue (MB) with bovine serum albumin (BSA) was carried out by spectroscopic methods. Denaturation curves and absorption and differential absorption spectra of the protein-ligand complexes were obtained. It was shown that the denaturation temperature of the albumin complexes of BSA with Hoechst 33258 decreases with increase of the ligand/protein concentration ratio whereas in the case of MB the denaturation temperature increases. Changes in the absorption spectra and differential absorption spectra of the complexes of the ligands with albumin resulting from combination of these specific ligands with the protein were observed. It is supposed that during interaction of Hoechst 33258 with BSA some loosening of the protein compact structure occurs as a result of partial loss of helicity in the α structures, whereas in the case of MB the degree of packing in the spatial structure of the protein is increased. [ABSTRACT FROM AUTHOR]

Interaction of the minor groove binder, Hoechst 33258, with the Dickerson-Drew DNA dodecamer sequence has been investigated using docking, MM/QM, MM/GBSA and molecular dynamics computations to study the modes of binding and the interactions responsible for the binding. Besides the original Hoechst 33258 ligand ( HT ), a total of 12 ionization and stereochemical states for the ligand are obtained at the physiological pH and have been docked into B-DNA. These states have one or the other or both benzimidazole rings in protonated states, apart from the piperazine nitrogen, which has a quaternary nitrogen in all the states. Most of these states are found to exhibit good docking scores and free energy of binding with B-DNA. The best docked state has been taken further for molecular dynamics simulations and compared with the original HT . This state is protonated at both benzimidazole rings besides the piperazine ring and hence has very highly negative coulombic interaction energy. In both cases, there are strong coulombic interactions, but these are offset by the almost equally unfavorable solvation energies. Thus, the nonpolar forces, particularly van der Waals contacts, dominate the interaction, and the polar interactions highlight subtle changes in the binding energies, leading to more highly protonated states having more negative binding energies.Communicated by Ramaswamy H. Sarma.

I summarize here the history of the use of abbreviations, mostly in subject areas related to dyes, stains and fluorescent probes used in biology and medicine. The dozen most popular abbreviations in these fields are identified and their salient characteristics noted. The pros and cons of each abbreviation are discussed with relevant citations. Certain abbreviations that are not in the list, e.g., AZAN and LN, are mentioned because they have an unusual origin; while others, i.e., INEPT and INADEQUATE are presented because they are bizarre. A related topic is abbreviations used for citations, which require further efforts to decipher. In the past, brevity helped conserve materials, such as ink and paper, and promoted more rapid publishing. I suggest that the use of many abbreviations in the current era of electronic publishing may not be necessary. [ABSTRACT FROM AUTHOR]

The flow cytometry‐based assay has been increasingly used to assess the cell‐mediated cytotoxicity since the 1980s due to its advantages over the conventional radioactive 51Cr release assay (CRA), such as higher sensitivity at the single‐cell level and nonradioactivity. The basic principle of this assay is the usage of two dyes, one nontoxic dye for labeling targets or effector cells to distinguish one from another, one viability dye for discrimination of dead from live cells. Due to the problem of spontaneous release or leakage of the nontoxic dye, the concern about the cross‐staining has not yet been clearly elucidated. In this study, carboxyfluorescein diacetate succinimidyl ester (CFSE) was utilized to label target cells and Hoechst 33258 was used as the viability dye. We confirmed that no cross‐staining occurred between the effector and target cells after 4 h of coculture. We also found that the cytotoxicity would be overestimated if effector cells instead of target cells were labeled due to the exclusion of viable targets in effector‐target conjugates. Using EDTA at the end of culture or labeling targets can solve this problem. Furthermore, the gating strategy could be improved by plotting CFSE against forward scatter (FSC) to discriminate some early apoptotic events. Due to the loss of target cells lysed by effector cells, counting beads are normally preferable in this assay. Here, we found an alternative to the use of beads in standardizing the flow cytometry‐based assay. Instead of using beads, sample acquisition in a fixed time was shown to have the same effect in specific lysis evaluation as the beads application but have a greater stability than the latter. With a good quality control, the acquisition time for each sample could be shortened to 15 s, thus making this work to be done efficiently, especially in the case of larger sample sizes. Collectively, the findings in this study can improve the flow cytometric cytotoxicity assay to be carried out in a more accurate, efficient, and cost‐effective way. © 2020 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

As the carrier of genetic information, the DNA double helix interacts with many natural ligands during the cell cycle, and is amenable to such intervention in diseases such as cancer biogenesis. Proteins bind DNA in a site-specific manner, not only distinguishing between the geometry of the major and minor grooves, but also by making close contacts with individual bases within the local helix architecture. Over the last four decades, much research has been reported on the development of small non-natural ligands as therapeutics to either block, or in some cases, mimic a DNA–protein interaction of interest. This review presents the latest findings in the pursuit of novel synthetic DNA binders. This article provides recent coverage of major strategies (such as groove recognition, intercalation and cross-linking) adopted in the duplex DNA recognition by small molecules, with an emphasis on major works of the past few years.

Thermodynamic analysis of Hoechst 33258 interaction with poly(rA)-poly(rU) at the ionic strengths of the solution 0.04, 0.02 and 0.1 M has been carried out. Values of both binding constant (K) and number of nucleotides (n) per binding site were determined from Scatchard’s curves that were obtained based on spectroscopic and electrochemical measurements. Values of changes of enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) were also determined at the complex-formation. It should be mentioned that the binding of Hoechst 33258 to nucleic acids is accompanied by positive change of enthalpy. Meanwhile depending on the ionic strength of the solution, the values of DH for DNA change insignificantly, while for poly(rA)-poly(rU) this parameter is exposed to relevant alterations. [ABSTRACT FROM AUTHOR]

The DNA hydrolysis by deoxyribonuclease (DNAse I) in aqueous solution was studied, using fluorescence spectroscopy and high-sensitive light-scattering detection. Specific hydrolysis of high-polymer DNA or fragmented DNA by the enzyme led to a strong decrease in the fluorescence of the Hoechst dye. The hydrolysis of mitochondrial DNA was accompanied by a decrease in the fluorescence of the dye only in 1.6 times. Hydrolysis within minutes and even hours led to appearance of large polynucleotide fragments, but not to short oligonucleotides, that was confirmed using polarized fluorescence and highly sensitive measurement of light-scattering. At the moment of the time of formation of a complex between DNA and DNAse I, a strong light-scattering occurred, which then dropped sharply during hydrolysis of high-molecular DNA, and slowly decreased during hydrolysis of fragmented DNA. The proposed methods can be applied for selective detection of trace amounts of various types of DNA, as well as for studying their physic-chemical properties. [ABSTRACT FROM AUTHOR]

Interaction of the important minor groove binder Hoechst 33258 with poly(A).poly(U) was studied using isothermal titration calorimetry. The equilibrium constant of the association was calculated to be (4.00 ± 0.80) 10 6  M −1 at 298.15 K. The Hoechst RNA binding was driven by large negative enthalpy contributions. The equilibrium constant values for the binding reaction decreased with increasing [Na + ] concentration. Parsing of the standard molar Gibbs energy change revealed that non-polyelectrolytic forces dominated the complexation process at all the salt concentrations studied. Temperature dependent calorimetric studies revealed that the equilibrium constant decreased in magnitude with increasing temperature in the range (288.15 ± 0.10)–(308.15 ± 0.10) K. Furthermore, there were significant alterations in the thermodynamic parameters with change in temperature; the binding became increasingly enthalpy driven and the standard molar entropic contribution decreased in magnitude and became unfavourable as the temperature was increased. Negative heat capacity change and an enthalpy-entropy compensation phenomenon were also observed. The data observed may be useful for developing structure selective RNA-based therapeutics. [ABSTRACT FROM AUTHOR]

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

The interaction of groove binding compounds — peptide antibiotic (polyamide) netropsin and fluorescent dye (bisbenzimidazole) Hoechst 33258 — with the double-stranded DNA and synthetic double-stranded polynucleotide poly(rA)-poly(rU) has been studied by spectrophotometry. Absorption spectra of these ligand complexes with nucleic acids have been obtained. Spectral changes at the complexation of individual ligands with the mentioned nucleic acids reveal the similarity of binding of each of these ligands with both DNA and RNA. Based on the spectroscopic measurements, the binding parameters of netropsin and Hoechst 33258 binding to DNA and poly(rA)-poly(rU) - K and n, as well as the thermodynamic parameters ΔS, ΔG, and ΔH have been determined. It was found that the binding of Hoechst 33258 to both nucleic acids is accompanied by a positive change in enthalpy, while in the case of netropsin the change in enthalpy is negative. Moreover, the contribution of entropy to the formation of the complexes is more pronounced in the case of Hoechst 33258. [ABSTRACT FROM AUTHOR]

A new complex, [Pt(valcyte)(DMSO)Cl]Cl, in which valcyte (trade name) served as valganciclovir hydrochloride drug ([2-[(2-amino-6-oxo-3H-purin-9-yl)methoxy]-3-hydroxypropyl](2S)-2-amino-3-methylbutanoate), was synthesized and characterized by different physicochemical methods. Binding interaction of this complex with calf-thymus DNA (ct-DNA) has been investigated by multispectroscopic techniques. The complex displays significant binding properties with ct-DNA. The results of fluorescence and UV-vis absorption spectroscopy indicated that this complex interacted with ct-DNA in a groove-binding mode, and the binding constant was 3.8 × 104 M−1. Furthermore, the complex induced detectable changes in the CD spectrum of ct-DNA and slightly changed its viscosity which verified the groove-binding mode. Finally, all results indicated that Pt(II) complex interact with DNA via groove-binding mode. [ABSTRACT FROM AUTHOR]

Background: Based on the pivotal role of astrocytes in brain homeostasis and the strong metabolic cooperation existing between neurons and astrocytes, it has been suggested that astrocytic dysfunctions might cause and/or contribute to neuroinflammation and neurodegenerative processes. Therapeutic approaches aimed at both neuroprotection and neuroinflammation reduction may prove particularly effective in slowing the progression of these diseases. The endogenous lipid mediator palmitoylethanolamide (PEA) displayed neuroprotective and anti(neuro)inflammatory properties, and demonstrated interesting potential as a novel treatment for Alzheimer's disease.Objective and Methods: We firstly evaluated whether astrocytes could participate in regulating the Aβ42-induced neuronal damage, by using primary mouse astrocytes cell cultures and mixed astrocytes-neurons cultures. Furthermore, the possible protective effects of PEA against Aβ42-induced neuronal toxicity have also been investigated by evaluating neuronal viability, apoptosis, and morphometric parameters.Results: The presence of astrocytes pre-exposed to Aβ42 (0.5μM; 24 h) induced a reduction of neuronal viability in primary mouse astrocytes-neurons co-cultures. Furthermore, under these experimental conditions, an increase in the number of neuronal apoptotic nuclei and a decrease in the number of MAP-2 positive neurons were observed. Finally, astrocytic Aβ42 pre-exposure induced an increase in the number of neurite aggregations/100μm as compared to control (i.e., untreated) astrocytes-neurons co-cultures. These effects were not observed in neurons cultured in the presence of astrocytes pre-exposed to PEA (0.1μM), applied 1 h before and maintained during Aβ42 treatment.Conclusion: Astrocytes contribute to Aβ42-induced neurotoxicity and PEA, by blunting Aβ42-induced astrocyte activation, improved neuronal survival in mouse astrocyte-neuron co-cultures. [ABSTRACT FROM AUTHOR]

It has been shown that intercalating (mitoxantone, doxorubicin) and non-intercalating (netropsin, Hoechst 33258) ligands form more strong complex with nucleic acids irradiated by resonant frequencies (64,5 and 50.3 GHz) for oscillations of water structures. Under the effect of the mentioned non-thermal millimeter waves a dehydration of nucleotides occurs, which results in increasing of the binding constant of ligands with irradiated double-stranded nucleic acids (NA). Calculations showed that as a result of irradiation, the enthalpy (ΔH) and entropy (ΔS) of the binding of ligands to NA has been changed. However the value of ΔH is mainly being changed for intercalators, and the value of ΔS - for non-intercalators. [ABSTRACT FROM AUTHOR]

Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil ( Br U)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to Br U caused DNA cleavage preferentially at self-complementary 5′-AA Br U Br U-3′ sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA. [ABSTRACT FROM AUTHOR]

In the present work, the interaction of Isoxsuprine (ISX) with Calf thymus DNA (ct-DNA) under physiological conditions (Tris–HCl buffer of pH 7.4) was investigated by using electronic absorption, circular dichroism, viscosity, electrochemical studies, fluorescence techniques, salt effect studies and computational studies. Competitive fluorimetric studies with Hoechst 33258 have shown that ISX exhibit the ability to displace the DNA-bound Hoechst 33258, indicating that it binds to ct-DNA in strong competition with Hoechst 33258 for the minor groove binding. Furthermore, the resulting data showed that ISX cannot displace methylene blue or acridine orange, which are the common intercalator molecules. The viscosity of ct-DNA solution was almost unchanged on addition of ISX and circular dichroism (CD) spectra of ct-DNA showed small changes in the presence of ISX which is in agreement with groove binding mode of interaction. Thus all above studies showed that the ISX drug binds to ct-DNA in a groove binding mode.The salt-effect studies showed the non-electrostatic nature of binding of ISX to ct-DNA. Moreover, molecular docking results support the above experimental data and suggest that ISX prefers to bind on the minor groove of ct-DNA. [ABSTRACT FROM AUTHOR]

Oxysophoridine (OSR) is a major active alkaloid extracted from Sophoraalopecuroides L. The aim of the present study was to investigate the induction of the apoptotic effects of OSR on colorectal cancer cells in vivo and in vitro. The results of the MTT and colony formation assays demonstrated that the proliferation of HCT116 cells was inhibited by OSR in vitro. The characteristics of cellular apoptosis in OSR‑treated HCT116 cells were analyzed by Hoechst 33258 staining. It was also observed that the expression of caspase‑3, B‑cell lymphoma‑2 (Bcl‑2) associated X protein (Bax) and cytochrome c increased significantly upon OSR treatment. However, the expression of Bcl‑2 and poly ADP‑ribose polymerase‑1 (PARP‑1) was downregulated in OSR‑treated cells compared with untreated cells. The in vivo experiments identified that OSR significantly inhibited the growth of the transplanted mouse CT26 tumor tissue, upregulated the expression of caspase‑3, Bax and cytochrome c and downregulated the expression of Bcl‑2 and PARP‑1, as detected by reverse transcription‑quantitative polymerase chain reaction and western blotting. It may be concluded that OSR significantly induced apoptotic effects on colorectal cancer cells in vivo and in vitro, and that its mechanism may be associated with the Bcl‑2/Bax/caspase‑3 signaling pathway. [ABSTRACT FROM AUTHOR]

Fragile sites represent regions of chromatin that fail to compact during mitosis. Based on the prevalence and pattern of inheritance they are classified as rare fragile sites or common fragile sites. Rare fragile sites either occur spontaneously or can be induced by certain ATspecific binding chemicals namely distamycin, Hoechst 33258, Berenil and others. The most common of all rare autosomal fragile sites is fra(16)(q22) with a heterozygote frequency of ~5%. FRA16B results from an expansion of a 33 bp AT-rich minisatellite repeat. These rare forms are usually heritable and segregate in a Mendelian fashion. The proband who was referred for secondary amenorrhoea, revealed 46,XX,fra(16)(q22.1)pat karyotype. Her father and younger sibling were also found to be carriers. This study aimed to delineate the genotypic and phenotypic features exhibited by these carriers and to evaluate FRA16B expression using AT-specific binding chemicals. The additives employed were Berenil, BrdU and Hoechst 33258. Berenil at a concentration of 150 µg/ml showed the highest expression of FRA16B. Although the recent breakthrough in molecular characterization of fragile sites plays a critical role in comprehending their association with various diseases, the physiological link between them and amenorrhoea is not clearly understood.

This study reports (for the first time) the staining ability of vital (0.4% trypan blue and 1% neutral red) and fluorescent (Hoechst 33258) dyes to differentiate between live and dead Schistosoma haematobium (S. haematobium) eggs in human urine samples. Since S. haematobium egg is important in disease pathology, diagnosis, transmission, and drug development research, it is essential to be able to easily distinguish live eggs from dead ones. Staining is considered a way of enhancing the identification of live and dead eggs. Urine samples from school children were examined for the presence of S. haematobium eggs. Vital and fluorescent dyes were used to stain the samples that contained S. haematobium eggs, after which they were observed using light and fluorescent microscopes, respectively. The Hoechst 33258 provided a good staining outcome for differentiation between live and dead eggs, followed by 0.4% Trypan blue. Regarding the 1% neutral red stain, even though it provided some evidence of which egg was alive or dead, the distinction was not very clear; therefore, it could be useful when used in combination with other stains for egg viability determination. The benefits of this study will include assessing the effect of drugs on S. haematobium eggs in Schistosomiasis research.

G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the neomycin and Hoechst 33258 units.

The binding of both ethidium bromide (EtBr) and Hoechst 33258 (H33258) to calf thymus DNA differing by the mechanism at different ionic strengths of NaCl water solutions (2, 20 and 150mM) has been quantitatively studied by square wave voltammetry (SWV). It was revealed that EtBr binds to DNA by more than one mode: at the solution ionic strengths 2 and 150mM, two modes were revealed, at 20mM - three modes. Values of EtBr binding constant () to DNA and the number of nucleotides per one binding site () for respective binding modes were determined. For H33258 under the same conditions, two binding modes were found and for both these modes values of and were determined. Dependence of the binding types and parameters on the solution ionic strength by SWV method was revealed, which differs by the binding type of EtBr and H33258. The obtained data are in good accordance with the ones determined by spectroscopic (absorption and fluorescence) and differential pulse voltammetry (DPV) methods. The obtained data indicate the sensibility and high precision of SWV method which may be applied to studies on interaction of different ligands with DNA, which reveal some peculiarities of their binding to DNA that are hidden and are not found by other methods. [ABSTRACT FROM AUTHOR]

The interaction of a novel macrocyclic copper(II) complex, ([CuL(ClO4)2] that L is 1,3,6,10,12,15-hexaazatricyclo[13.3.1.16,10]eicosane) with calf thymus DNA (ct-DNA) was investigated by various physicochemical techniques and molecular docking at simulated physiological conditions (pH = 7.4). The absorption spectra of the Cu(II) complex with ct-DNA showed a marked hyperchroism with 10 nm blue shift. The intrinsic binding constant (Kb) was determined as 1.25 × 104M−1, which is more in keeping with the groove binding with DNA. Furthermore, competitive fluorimetric studies with Hoechst33258 have shown that Cu(II) complex exhibits the ability to displace the ct-DNA-bound Hoechst33258 indicating that it binds to ct-DNA in strong competition with Hoechst33258 for the groove binding. Also, no change in the relative viscosity of ct-DNA and fluorescence intensity of ct-DNA-MB complex in the present of Cu(II) complex is another evidence to groove binding. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrogen bonds and van der Waals interactions played major roles in the binding reaction. The experimental results were in agreement with the results obtained via molecular docking study. [ABSTRACT FROM PUBLISHER]

Drugs which are capable of targeting sequence specific DNA constitute a novel approach to chemotherapy and have been exploited in detail in recent years. In this context, the synthetic minor groove binder Hoechst 33258 has been in focus due to its ability to specifically bind double helical DNA. A theoretical study of the structure, stability and properties of the tautomers and rotamers of Hoechst 33258 in two different dielectric media, viz. gas phase and aqueous solution would be useful in this respect. Besides these studies, the electronic, vibrational, 1 H NMR and 13 C NMR spectra are also investigated in detail in this work. These are also compared with the experimental spectra. The results indicate that in the gas phase, in all these rotamers, while the three aromatic rings prefer the same plane, the piperazinyl ring projects out of plane. The degree of non-planarity of the central bond increases in aqueous medium. The molecule retains its aqueous state conformation on binding to DNA. Bond length, bond order and NBO analyses suggest an enhanced degree of conjugation in the bonds connecting the three aromatic rings. [ABSTRACT FROM AUTHOR]

Monomeric bis-benzimidazole (MB) is an analog of the Hoechst 33258 dye. The enthalpy and entropy of MB binding were evaluated by analyzing the calorimetric data on MB reverse titration with poly(dA) · poly(dT). A mathematical model was developed to estimate the thermodynamic parameters of binding on the basis of calorimetric data. The results agree well with spectrophotometric data on the binding of analogous compounds. The model was used to estimate the parameters of binding with poly(dA) · poly(dT) for dimeric bis-benzimidazole (DB), which consists of two bis-benzimidazole monomers linked via a flexible chain. The ligand was assumed to produce different types of complexes with the polymer. [ABSTRACT FROM AUTHOR]

Neomycin and Hoechst 33258 are two well-known nucleic acid binders that interact with RNA and DNA duplexes with high affinities respectively. In this manuscript, we report that covalent attachment of bisbenzimidazole unit derived from Hoechst 33258 to neomycin leads to intercalative binding of the bisbenzimidazole unit (oriented at 64–74° with respected to the RNA helical axis) in a linker length dependent manner. The dual binding and intercalation of conjugates were supported by thermal denaturation, CD, LD and UV–Vis absorption experiments. These studies highlight the importance of linker length in dual recognition by conjugates, for effective RNA recognition, which can lead to novel ways of recognizing RNA structures. Additionally, the ligand library screens also identify DNA and RNA selective compounds, with compound 9 , containing a long linker, showing a 20.3 °C change in RNA duplex T m with only a 13.0 °C change in T m for the corresponding DNA duplex. Significantly, the shorter linker in compound 3 shows almost the reverse trend, a 23.8 °C change in DNA T m , with only a 9.1 °C change in T m for the corresponding RNA duplex. [ABSTRACT FROM AUTHOR]

In the present work Förster resonance energy transfer (FRET) between DNA-specific fluorescent dyes Hoechst 33258 (Ht58; donor) and SYBR Green I (SG; acceptor) is compared in isotropic DNA solution and in its cholesteric liquid-crystalline dispersion (DNA CLCD). At a fixed donor concentration of 3.57 × 10−6 М in the spatially ordered system, FRET starts at lower acceptor concentration than in the isotropic system and at the SG concentration below 1.05 × 10−7 M, its efficiency in DNA CLCD is up to ≈4-fold higher than in DNA solution (3D-FRET vs. 1D-FRET). However, the maximum FRET efficiency is higher in isotropic solution (≈70% vs. ≈50%, respectively) apparently due to the absence of steric restrictions on intermolecular interactions imposed by the dense packaging of the DNA molecules. In isotropic DNA solution FRET from Ht58 to SG is accompanied by the displacement of Ht58 from the minor-groove complex as the SG concentration increased, while the displacement of Ht58 in DNA CLCD seems to be limited. Using FRET between Ht58 and SG, the formation of DNA CLCD particles via stopped-flow method was investigated and for the first time the subsecond stage of the DNA CLCD particle formation, generation of "precholesteric" optically inactive nuclei, was demonstrated. • FRET from Hoechst 33258 to SYBR Green I was studied in isotropic and crowded DNA. • In crowded DNA FRET started earlier but was limited by steric restrictions. • Maximum FRET efficiency was ≈50% in crowded DNA and ≈70% in DNA solution. • FRET was used to study the formation of DNA CLCD particles. • Subsecond stage of optically inactive nuclei formation was shown for the first time. [ABSTRACT FROM AUTHOR]

Praziquantel (PZQ) is effective against all the evolutive phases of Schistosoma mansoni. Infected Biomphalaria glabrata snails have their cercarial shedding interrupted when exposed to PZQ. Using primary in vitro transformed sporocysts, labeled with the probe Hoechst 33258 (indicator of membrane integrity), and lectin of Glycine max (specific for carbohydrate of N-acetylgalactosamine membrane), we evaluated the presence of lysosomes at this evolutive phase of S. mansoni, as well as the influence of PZQ on these acidic organelles and on the tegument of the sporocyst. Although the sporocyst remained alive, it was observed that there was a marked contraction of its musculature, and there occurred a change in the parasite's structure. Also, the acidic vesicles found in the sporocysts showed a larger delimited area after contact of the parasites with PZQ. Damages to the tegument was also observed, as show a well-marked labeling either with Hoechst 33258 or with lectin of Glycine max after contact of sporocysts with the drug. These results could partially explain the interruption/reduction mechanism of cercarial shedding in snails exposed to PZQ.

The rise and emergence of resistance to antifungal drugs by diverse pathogenic fungal strains have resulted in an increase in demand for new antifungal agents. Various heterocyclic scaffolds with different mechanisms of action against fungi have been investigated in the past. Herein, we report the synthesis and antifungal activities of 18 alkylated mono-, bis-, and trisbenzimidazole derivatives, their toxicities against mammalian cells, as well as their ability to induce reactive oxygen species (ROS) in yeast cells. Many of our bisbenzimidazole compounds exhibited moderate to excellent antifungal activities against all tested fungal strains, with MIC values ranging from 15.6 to 0.975 μg/mL. The fungal activity profiles of our bisbenzimidazoles were found to be dependent on alkyl chain length. Our most potent compounds were found to display equal or superior antifungal activity when compared to the currently used agents amphotericin B, fluconazole, itraconazole, posaconazole, and voriconazole against many of the strains tested. [ABSTRACT FROM AUTHOR]

Rhein is an active component of rhubarb; a traditional Chinese medicine reported to induce apoptosis and cause liver toxicity. However, rhein's apoptotic-inducing effects, as well as its molecular mechanisms of action on hepatic cells need to be further explored. In the present study, rhein was found to trigger apoptosis in primary human hepatic HL-7702 cells as showed by annexin V/PI double staining assay and nuclear morphological changes demonstrated by Hoechst 33258 staining. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was caspase-dependent, presumably via ER-stress associated pathways, as illustrated by up-regulation of glucose-regulated protein 78 (GRP 78), PKR-like ER kinase (PERK), C-Jun N-terminal kinase (JNK) and CCAAT/enhancer-binding protein homologous protein (CHOP). Meanwhile, caspase-4 as a hallmark of ER-stress, was also showed to be activated following by caspase-3 activation. Furthermore, rhein also promoted intracellular elevation of calcium that contributed in apoptosis induction. Interestingly, pre-treatment with calpain inhibitor I reduced the effects of rhein on apoptosis induction and JNK activation. These data suggested that rhein-induced apoptosis through ER-stress and elevated intracellular calcium level in HL-7702 cells. [ABSTRACT FROM AUTHOR]

The study of EtBr and H33258 interaction as well as EtBr and MB interaction with DNA has been carried out. It was revealed that, at joint interaction, the effect of two ligands on the change of melting thermodynamic parameters of EtBr-DNA-H33258 or EtBr-DNA-MB complexes is not an addition of separate interaction influences. It was shown that, at joint, binding of EtBr and MB with DNA competition occurs, while in the case of EtBr and H33258, the mutual strengthening of stabilizing effect of each of them on DNA double-stranded structure mainly takes place. [ABSTRACT FROM AUTHOR]

The design, synthesis, and DNA binding properties of aza Hx-PI or p -anisyl-4- aza -benzimidazole-pyrrole-imidazole ( 5 ) are described. Aza Hx, 2-( p -anisyl)-4- aza -benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, Δ T M , and SPR studies provided evidence that an aza Hx/IP pairing, formed from antiparallel stacking of two aza Hx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C–G doublet. Aza Hx-PI was found to target 5′-ACGCGT-3′, the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity ( K eq 4.0 ± 0.2 × 10 7 M −1 ). [ABSTRACT FROM AUTHOR]

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study. [ABSTRACT FROM AUTHOR]

Hoechst 33258 derivatives with additional interacting moieties attached at the ends of branched linkers were synthesized, and their DNA binding properties were investigated with regard to the A3T3 repeat by measuring fluorescence spectra. The binding property of the ligand was investigated by fluorescence titration, and the titration data were analyzed using the McGhee–von Hippel method. Ligand 6Q with the quinolin-6-yloxyacetyl group and Ligand IQ with isoquinolin-6-yloxyacetyl group at the ends of the branched linkers exhibit highly positive cooperativity for the DNA having 5 A3T3 sites with 3 base-insertions between them with sequence selectivity. The strategy developed in this study may be generally applicable for designing ligands for repetitive DNA sequences. [ABSTRACT FROM AUTHOR]

Spectrophotometric study of Hoechst 33258 binding to DNA has been investigated. It was shown that this ligand forms at least two types of complexes at 0.002 M and 0.02M ionic strengths of solution. One of them is strong, the other is weak. It was revealed that at 0.02M ionic strength of solution, the binding constant value by strong mode is one order higher than at 0.002M. It was obtained that at both ionic strengths of solution at weak mode of interaction the values of binding constant of Hoechst 33258 to DNA coincide with each other. [ABSTRACT FROM AUTHOR]

Extracellular ATP is known to permeabilize certain cell types to polyatomic cations like YO-PRO1. Here, we report that extracellularly applied ATP stimulated rapid uptake and accumulation of an otherwise weakly membrane permeable fluorescent DNA-binding cytotoxin, Hoechst 33258, into cervical cancer cells. While ATP stimulated Hoechst 33258 uptake in 20–70% of cells from seven cervical cancer cell lines, it stimulated uptake in less than 8% of cervical epithelial cells obtained from the normal transformation zone and ectocervix tissue of 11 patients. ATP-evoked Hoechst 33258 uptake was independent of ionotropic P2X receptors, but dependent on activation of P2Y receptors. Thus, we show here that cervical cancer cells can be selectively induced to take up and accumulate an ionic cytotoxin by exposure to extracellular ATP. [ABSTRACT FROM AUTHOR]

In this study, we synthesized a Hoechst 33258–conjugated hyaluronated fullerene consisting of Hoechst 33258 (as a target moiety to detect necrotic tumor cells), hyaluronic acid (as a target polymer to bind the CD44 receptor overexpressed on the surface of tumor cells), and fullerene (as a photosensitizing agent). This conjugate self-assembled to form nanoparticles consisting of a hydrophilic block (Hoechst 33258 and hyaluronic acid) and a lipophilic block (fullerene). We utilized these nanoparticles to improve the antitumor efficacy via photodynamic tumor therapy. The HCT-116 cells that were damaged after the first photodynamic tumor therapy (using hyaluronated fullerene nanoparticles) were again targeted using Hoechst 33258–conjugated hyaluronated fullerene nanoparticles (detecting necrotic tissues). The experimental results revealed that the second photodynamic tumor therapy using Hoechst 33258–conjugated hyaluronated fullerene nanoparticles caused significant increases in the in vitro phototoxicity and the in vivo tumor inhibition, thereby suggesting their pharmaceutical potential for designing effective multiple photodynamic tumor therapy treatments. [ABSTRACT FROM PUBLISHER]