Thermodynamic Study of Rhodamine 123-Calf Thymus DNA Interaction: Determination of Calorimetric Enthalpy by Optical Melting Study

In this paper, the interaction of rhodamine123 (R123) with calf thymus DNA has been studied using molecular modeling and other biophysical methods like UV–vis spectroscopy, fluoremetry, optical melting, isothermal titration calorimetry, and circular dichroic studies. Results showed that the binding energy is about −6 to −8 kcal/mol, and the binding process is favored by both negative enthalpy change and positive entropy change. A new method to determine different thermodynamic properties like calorimetric enthalpy and heat capacity change has been introduced in this paper. The obtained data has been crossed-checked by other methods. After dissecting the free-energy contribution, it was observed that the binding was favored by both negative hydrophobic free energy and negative molecular free energy which compensated for the positive free energies due to the conformational change loss of rotational and transitional freedom of the DNA helix

<i>In</i><i>vivo</i> efficacy of

<p>HM. BALB/c mice were fed with HM (0.25 or 0.5 mg/kg) or ACV (5 mg/kg) and after 8 h of drug treatment the animals were infected with HSV-2G (9 X 10⁵ pfu per animal) intravaginally. The challenged animals of test groups were fed with HM twice daily for 7 days. Development of lesions and death were observed three times daily, while brain and vaginal tissue were collected after sacrification on days 2, 4, 6 or 8 after infection, homogenized and centrifuged. The supernatant was used for the determination of virus yield by plaque assay. Mean lesion score [A], Mean±S.D. of virus yield at log₁₀ (PFU/organ) in vaginal tissue [B] and brain [C]. </p

Anti-HSV efficacy of HM.

<p>[A] <b>Plaque </b><b>Reduction </b><b>Assay</b>. Infected cells were treated with HM or ACV at 0.5-50 μg/ml, overlaid with methylcellulose and plaques developed after 2-3 days were stained. The % of plaque number reduction was calculated, and the effective concentration of drug that inhibited the number of viral plaques was interpolated from the dose-response curve. [B] <b>Time </b><b>course </b><b>analysis</b>. Inhibitory effects of HM and ACV at various time points prior to infection (-3 to -1 h), at the time of infection (0 h) and post-infection (2-24 h) with HSV-2 were evaluated by plaque reduction assay. Each bar represents the mean ± S.E.M of three independent experiments.</p

Histopathology of Genital tissue of Balb/C mice

<p>: uninfected [A], infected with HSV-2G [B], HSV-2G infected animals treated with 0.5 µg/ml of HM [C], and ACV at 5 µg/ml [D]. </p

Effect of HM on viral IE transcriptional events.

<p>[A] <b>EMSA</b>: HSV-2G infected Vero cells were treated with HM for 2 and 4 h and assayed for EMSA. Biotin-labelled oligo was present in Lanes 1-6; P, biotin labelled oligo, M, mock control. [B] <b>Supershift </b><b>assay</b>: nuclear extracts from HSV-2G infected HM treated cells for 4 h p.i. was pre-incubated with HCF-1 polyclonal antibodies, added with reaction mixture, applied to non-denaturing 4% polyacrylamide gels and visualized by autoradiography. P, free biotin labelled probe; ns, nonspecific binding. [C] HCF-1 or LSD1 were immunoprecipitated in HM treated virus infected Vero cell lysate and the association was confirmed by immunoblotting with anti-HCF-1 and anti-LSD1 antibodies. </p