In silico investigation of DNA minor groove binding bibenzimidazoles in the context of UVA phototherapy.

The versatility of DNA minor groove binding bibenzimidazoles extends to applications in cancer therapy, beyond their typical use as DNA stains. In the context of UVA phototherapy, a series of halogenated analogues designated ortho-, meta-, and para-iodoHoechst have been investigated. Phototoxicity involves dehalogenation of the ligands following exposure to UVA light, resulting in the formation of a carbon-centred radical. While the cytotoxic mechanisms have been well established, the nature and severity of DNA damage induced by the ortho-, meta-, and para-iodoHoechst isomers requires clarification. Our aims were to measure and compare the binding constants of iodoHoechst analogues, and to determine the proximity of the carbon-centred radicals formed following photodehalogenation to the C1', C4', and C5' DNA carbons. We performed molecular docking studies, as well as classical molecular dynamics simulations to investigate the interactions of Hoechst ligands with DNA including a well-defined B-DNA dodecamer containing the high affinity AATT minor groove binding site. Docking highlighted the binding of Hoechst analogues to AATT regions in oligonucleotides, nucleosomes, and origami DNA helical bundles. Further, MD simulations demonstrated the stability of Hoechst ligands in the AATT-containing minor groove over microsecond trajectories. Our findings reiterate that the efficiency of dehalogenation per se, rather than the proximity of the carbon-centred radicals to the DNA backbone, is responsible for the extreme phototoxicity of the ortho- isomer compared to the meta- and para-iodoHoechst isomers. More generally, our analyses are in line with the potential utility of ortho-iodoHoechst in DNA-targeted phototherapy, particularly if combined with a cell-specific delivery system.

Selective Inhibition of Escherichia coli RNA and DNA Topoisomerase I by Hoechst 33258 Derived Mono- and Bisbenzimidazoles.

A series of Hoechst 33258 based mono- and bisbenzimidazoles have been synthesized and their Escherichia coli DNA topoisomerase I inhibition, binding to B-DNA duplex, and antibacterial activity has been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA topoisomerase I inhibition properties with IC50 values <5.0 µM. Several bisbenzimidazoles (3, 6, 7, 8) also inhibit RNA topoisomerase activity of E. coli DNA topoisomerase I. Bisbenzimidazoles inhibit bacterial growth much better than monobenzimidazoles for Gram-positive strains. The minimum inhibitory concentration (MIC) was much lower for Gram positive bacteria (Enterococcus spp. and Staphylococcus spp., including two MRSA strains 0.3-8 µg/mL) than for the majority of Gram negative bacteria (Pseudomonas aeruginosa, 16-32 µg/mL, Klebsiella pneumoniae > 32 µg/mL). Bisbenzimidazoles showed varied stabilization of B-DNA duplex (1.2-23.4 °C), and cytotoxicity studies show similar variation dependent upon the side chain length. Modeling studies suggest critical interactions between the inhibitor side chain and amino acids of the active site of DNA topoisomerase I.

An analysis method for simultaneous screening of deoxyribonucleic acid-binding active compounds and investigating their mechanisms by ultra-fast liquid chromatography tandem mass spectrometry coupled with fluorescence detection technology.

DNA has been known as the cellular target for many cytotoxic anticancer agents over the years. Discovering DNA-binding compounds has become an active research area, while various DNA-binding mechanisms make the drug discovery even more difficult. In this article, we present a novel analysis method to rapidly identify specific DNA-binding compounds from Pyrrosia lingua (Thunb.) using DNA-dual-fluorescent probes, ethidium bromide and Hoechst 33258, with the technology of ultra-fast liquid chromatography-diode array detector-tandem mass spectrometry and dual-wavelength fluorescence detector (UFLC-DAD-MS(n)-DFLD). Sixty-two compounds were identified, of which 22 were found to be active in DNA-binding. After investigation of their dose-response behaviors and structure-activity relationships, chlorogenic acids and flavonoid glycosides were found to be DNA-binders via both minor groove-binding and intercalation modes. The precision, reproducibility and stability of this method were validated by vitexin. The established system was sensitive, precise, and reliable to be used for both screening of DNA-binding compounds and investigating of their mechanisms.

Neuroprotective effect of Rhizoma Atractylodis macrocephalae against excitotoxicity-induced apoptosis in cultured cerebral cortical neurons.

Excitotoxicity has been implicated in neurological disorders. This study investigated the neuroprotective effect of the extract from Rhizoma Atractylodis macrocephalae on excitotoxicity-induced neuronal apoptosis in primary cultured cerebral cortical neurons. Excitotoxicity was induced by exposure of cortical neurons to glutamate. Neuronal apoptosis and the protective effect of Rhizoma Atractylodis macrocephalae extract were examined by multi-indices including cell viability assay, morphological features, DNA fragmentation and flow cytometric analysis. After exposure of cultured neurons to glutamate for 24 h, the neurons exhibited marked apoptotic-like death. Co-treatment of the neurons with glutamate and Rhizoma Atractylodis macrocephalae extract significantly elevated the cell viability, and reduced the number of apoptotic cells. These results demonstrate that Rhizoma Atractylodis macrocephalae is an effective neuroprotective agent against glutamate-induced excitotoxicity and may have therapeutic potential in excitotoxicity-mediated diseases.

Evaluation of osteoclastic resorption activity using calcium phosphate coating combined with labeled polyanion.

Osteoclasts are involved in bone resorption, and its activation is considered one of the causes of osteoporosis. The pit assay is the principal method for evaluating osteoclast function by measuring hydroxyapatite resorption in vitro. However, the pit assay requires time and trained techniques, including the pit image analysis, and there is no other easy method for evaluating bone resorption. In this study, we developed a novel approach to quantify the bone resorption activity using a calcium phosphate (CaP) coating labeled with fluorescent polyanion. Fluoresceinamine-labeled chondroitin polysulfate or Hoechst 33258-labeled deoxyribonucleic acid was used for CaP labeling. When macrophage cell line RAW264 was cultured on the labeled CaP under the stimulation with the receptor activator of the NF-κB ligand (RANKL), RAW264 cells differentiated into osteoclastic cells and the fluorescence intensity of the culture supernatant and pit area increased in a time- and dose-dependent manner. Furthermore, drugs for osteoporosis treatment, such as pamidronate and ß-estradiol, inhibited fluorescein release by the cells stimulated with RANKL. A positive correlation between the fluorescence intensity and pit area was observed (r=0.917). These results indicated that this new method using fluorescent polyanion-labeled CaP is a standardized useful assay system for the evaluation of bone resorption activity.

[Afferent nerve connection among "Hegu" (LI4), "Neiguan" (PC6), "Futu" (LI18) and thyroid gland region: fluorescent double labelling method].

OBJECTIVE: To observe the peripheral and central neural connection of acupoint "Hegu" (LI4), "Neiguan" (PC6), "Futu" (LI18) and the thyroid gland (TG) region with fluorescence double-labelling method. METHODS: Thirty male Wistar rats were divided into LI4-LI18 group, PC6-LI18 group, TG-LI 18 group, LI 4-TG group, and PC 6-TG group, with 6 rats in each. Fluorescence dyes Propidium Iodide (PI, 10 microL) and Bisbenzimide (Bb, 10 microL) were, separately, injected into those acupoints mentioned above and the TG region. Sixty hours after PI-injection and 12 hours after Bb-injection, the rats under deep anesthesia were transcardiacally perfused with PBS containing 4% polyoxymethylene, followed by taking the spinal cord and dorsal root ganglia (DRGs) of the cervical segments (C1-C8) and thoracic 1 (T1) segment. Fluorescent single- and dual-labeled cells of the sliced DRGs and cervical spinal cord were observed by fluorescence microscope. RESULTS: (1) All PI- and Bb-labeled cells were found to distribute in DRGs from C1-T1 segments. PI single-labeled cells from LI4 and PC6 mainly distributed in DRGs from C4 to C8. Bb single-labeled cells from LI18 and TG region distributed in DRGs from C1-C6. (2) Dual-labeled cells in the LI 4-LI 188 group, PC6-LI18 group, LI4-TG group, and PC6-TG group distributed in DRGs from C3 to C7, suggesting bifurcate peripheral processes of the cervical DRG neurons to innervate LI8, LI4, PC6 and the TG region. And the dual-labeled cells of the TG-LI 18 group distributed mainly in DRGs from C1 to C4. (3) A small number of single-labeled neurons(about 8% of total labeled cells in DRGs) and only several dual-labeled neurons were found in the anterior horn of the cervical spinal cord. CONCLUSION: LI18, LI4 and PC6 and the thyroid gland have the same peripheral cells in DRGs of C3-C7 segments, suggesting that the bifurcate peripheral innervation may provide an anatomic evidence for the analgesic effect of acupuncture of LI18, LI4 and PC6 on thyroidectomy.

Hit to lead account of the discovery of bisbenzamide and related ureidobenzamide inhibitors of Rho kinase.

A highly selective series of bisbenzamide inhibitors of Rho-associated coiled-coil forming protein kinase (ROCK) and a related ureidobenzamide series, both identified by high throughput screening (HTS), are described. Details of the hit validation and lead generation process, including structure-activity relationship (SAR) studies, a selectivity assessment, target-independent profiling (TIP) results, and an analysis of functional activity using a rat aortic ring assay are discussed.

[Effects of EGb 761 on the cell apoptosis induced by H2O2 in RIN-m beta cells].

OBJECTIVE: To investigate the effects of ginkgo biloba extract (EGb 761) on apoptosis induced by hydrogen peroxide (H2O2) in RIN-m beta-cells. METHODS: The apoptotic model was made by H2O2 exposed for six hours with a concentration of 500 micromol/L The cytotoxicity was measured by MTT. Hoechst 33258 fluorescent staining were used to detect the protective effect of EGb 761 on the apoptosis of RIN-m beta-cells induced by H2O2. Annexin V-PI double staining of Flow cytometry were used to detect apoptosis quantitively. RESULTS: Compare to control group, after exposed to 500 micromol/L H2O2 for 6 hours, the apoptosis rate incereased and cell survival rate were decreased considerably (P < 0.01). Pretreated for 10 hours with EGb 761, the flow cytometry results showed that the apoptosis rate decreased and cell survival rate were increased considerably (P < 0.01, compared to H2O2 control group). CONCLUSION: EGb 761 can decrease RIN-m beta-cells damage and apoptosis induced by H2O2.

DNA targeted UVA photosensitization: characterization of an extremely photopotent iodinated minor groove binding DNA ligand.

Previous studies have described UVA-induced DNA strand breakage at the binding sites of iodinated DNA minor groove binding bisbenzimidazoles. The DNA breakage, presumably mediated by the carbon-centred ligand radical produced by photodehalogenation, was also shown to be cytotoxic. The earlier studies included a comparison of three ligand isomers, designated ortho-, meta- and para-iodoHoechst, and the efficiency of photo-induction of strand breaks in plasmid DNA proved to be much higher for the ortho-isomer. We have now extended the comparison of the three isomers with respect to photo-induced cytotoxicity in K562 cells. Although the relationship between the extent of nuclear uptake and the concentration of the ligand in the medium was similar for the three isomers, assay of in situ dehalogenation in drug-treated cells indicated that the apparent cross-section for dehalogenation of the ortho-isomer was greater than 5-fold higher than that for the meta- and para-isomers. Also, analysis of clonogenic survival data showed that the dehalogenation event associated with ortho-iodoHoechst was a more efficient mediator of UVA-induced cytotoxicity in K562 cells than that for meta- or para-iodoHoechst. The number of dehalogenation events associated with 50% cell-kill for ortho-iodoHoechst (1.23+/-0.04 x 10(4)) was less than that for the para- (3.92+/-0.29 x 10(4)) and meta- (11.6+/-0.90 x 10(4)) isomers. Thus it is concluded that the photopotency of ortho-iodoHoechst, which is an important feature in the context of its potential use in clinical phototherapy, is due not only to more efficient UVA-mediated dehalogenation of the ligand, but also to greater cytotoxic potency per dehalogenation event.

Comparative investigation of the biocompatibility of various silicon nitride ceramic qualities in vitro.

There is a controversy about the biocompatibility of silicon nitride ceramics contained in the literature, which appears to be related to a factor of the individual chemical composition of different qualities of silicon nitride ceramics and of the different surface properties. This study attempts to investigate the cytotoxicity of different qualities of industrial silicon nitride ceramics applying an L929-cell culture model in a direct contact assay combined with a cell viability assessment. Five different qualities of industrial standard silicon nitride ceramics were chosen for in vitro testing. The chemical composition was determined by EDS analysis. Different biomedically approved aluminium oxide qualities, a titanium alloy, glass and polyvinylchloride (PVC) served as control materials. L929 mice fibroblasts were incubated directly on the materials for 24 h, stained with bisbenzimide and propidium iodine for double fluorochromasia viability testing, and evaluated by inversion-fluorescence microscopy to control cell morphology, viability and cell counts compared to empty well values. Scanning electron microscopy was applied to additionally investigate cell morphology. There was no observation of cytotoxic effects on the silicon nitride ceramic samples; moreover cell morphology remained the same as on aluminium oxide and titanium. Viability testing revealed the presence of avital cells exclusively on PVC, which served as a negative control. Cell counts on all polished surfaces showed significantly higher numbers, whereas some rough surface samples showed significantly lower numbers. We conclude that silicon nitride ceramics show no cytotoxic effects and should be considered for biomedical application owing to its favourable physiochemical properties, especially its superior resistance to mechanical stress, which would be useful for compression loaded conditions. Polished surfaces would appear to promote advanced biocompatibility.

Loosening of a preprophase band of microtubules in onion (Allium cepa L.) root tip cells by kinase inhibitors.

Effects of kinase inhibitors on the preprophase band of microtubules in onion (Allium cepa L.) root tip cells were examined. Bundled microtubules in preprophase bands were dispersed on the cell cortex when onion seedlings were incubated with 2.5-5.0 mM 6-dimethylaminopurine. Fifteen min was enough for the bundled microtubules to disappear. Although many preprophase bands remained when the seedlings were incubated with 60 microM staurosporin, these preprophase band microtubules were loosened and the width of the band became broad. These results sugget that some kinases are involved in the microtubule bundling in the preprophase band development.

DNA-drug interaction measurements using surface plasmon resonance.

The interactions of the drugs 2,7-bis[(diethylamino)-ethoxy]-fluoren-9-one dihydrochloride (Tilorone), 2,7-bis[(dipropylamino)-acetamido]-fluoren-9-one dihydrochloride (FA-2), 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride (Hoechst 33258), and hematoporphyrin IX derivative (HPD) with synthetic self-complementary DNA (36-b.p.; 5'-biotin-spacer-[d(CGCTATATAGCG)]3-3') were studied by SPR (Surface Plasmon Resonance). Monolayers of biotinylated DNA were immobilized on a streptavidin-dextran-gold triple-layer. Small portions of the drugs (approximately 30 pmol/ml) were injected in continuous flow. The mass corresponded to the amount of the bound molecules. Injections of 50 mM sodium hydroxide pulses separated the DNA double strands, releasing the effector molecules. Subsequent treatments with the effectors gave reproducible results. The maximum interaction between drug and DNA was observed in the case of Tilorone. 41 molecules could bind to the 36-b.p. DNA duplex. To investigate the microscopic behavior in condensed nucleic acid phases, SFM (Scanning Force Microscopy)-imaging and polarizing microscopic observations of DNA-effector complexes were carried out. Supplementary UV-absorption thermal denaturation curves of DNA with the above-mentioned effectors in dilute solutions were measured. As an additional aid to understand the geometries of DNA-drug interactions, computer simulations were performed and compared with the experimental data.

Lipid interference with fluorometric assay of DNA in adipose tissues under various conditions.

Interference by lipids with fluorometric assay of DNA in adipose tissues using Hoechst 33258 was investigated. Mixed glycerides shifted the emission maximum of standard DNA and induced a dose-dependent increase in fluorescence intensity. Glycerides in the samples containing a known concentration of DNA yielded erroneously higher DNA concentrations. The DNA concentrations obtained from acetone-defatted white and brown adipose tissues (WAT and BAT) were lower than those of non-defatted ones, while DNA content did not differ in low lipid-containing skeletal muscle between defatted and non-defatted samples, indicating that large amounts of lipids interfere with DNA measurement using Hoechst 33258 and that acetone defatting is a simple method to avoid this interference. Using this defatting method, the cellularity of WAT and BAT was estimated in rats under various experimental conditions. Cold-acclimation and repetitive immobilization stress decreased the body weight gain and the epididymal WAT weight. Sucrose overfeeding increased WAT weight but not body weight. These treatments of 4 weeks' duration did not induce any significant difference in WAT cell number from controls, while cold-acclimation increased the tissue cell number as well as the BAT weight.

Nuclease activity of 1,10-phenanthroline-copper. New conjugates with low molecular weight targeting ligands.

The chemical nuclease activity of 1,10-phenanthroline-copper depends on DNA sequence because the coordination complex has affinity for DNA. In order to target this efficient nucleolytic activity, it is essential to override its inherent specificity. The minimal size of ligands capable of redirecting the specificity has been investigated. A conjugate (HOP) prepared by alkylating Hoechst dye 33258 with 5-(iodoacetamido)-1,10-phenanthroline has a greater preference for A-T-rich regions than the unsubstituted 1,10-phenanthroline-copper complex, reflecting the specificity of this A-T-specific minor-groove binder. However, since quaternizing the dye with 5-(iodoacetamido)-1,10-phenanthroline increases its affinity for DNA, the specificity of cleavage by the conjugate is less than the binding selectivity of the dye. Linking 1,10-phenanthroline with the peptide of the helix-turn-helix domain of the Trp repressor specificity results in a conjugate with greater reactivity for the operator sequence than the unsubstituted complex. The intrinsic affinity of the 1,10-phenanthroline-Cu can only be partially overridden by the conformationally unstable peptide. Attachment of 1,10-phenanthroline to a deoxyoligonucleotide complementary to a single-stranded loop of RNA successfully targets the scission of the chemical nuclease. Cleavage sites are observed not only contiguous to the site of hybridization but also at nonadjacent sequence positions. The latter set of sites must be close in space to the 5' end of the hybridized deoxyoligonucleotide.

Binding of Hoechst 33258 and 4',6'-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents.

Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 10(8) (duplex M)-1 at 5.5 degrees C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence (Pjura et al., 1987; Teng et al., 1988; Larsen et al., 1989).