DNA minor groove binding of a well known anti-mycobacterial drug dapsone: A spectroscopic, viscometric and molecular docking study.

Dapsone is a sulfone drug mainly used as anti-microbial and anti-inflammatory agent for the treatment of various diseases including leprosy. Recently, its interaction with protein (bovine serum albumin) is evidenced. But, the binding propensity of this anti-mycobacterial drug towards DNA is still unknown. Also, the mode of dapsone-DNA interaction (if any) is still an unknown quantity. In this study, we have taken a thorough attempt to understand these two unknown aspects using various biophysical and in silico molecular docking techniques. Both UV-visible and fluorescence titrimetric studies indicated that dapsone binds to CT-DNA with a binding constant in order of 104 M-1. Circular dichroism, thermal denaturation and viscosity experiments revealed that dapsone binds to the grooves of CT-DNA. Competitive DNA binding studies clearly indicated the minor groove binding property of this anti-mycobacterial drug. Molecular docking provided detailed information about the formation of hydrogen bonding in the dapsone-DNA complex. This in silico study further revealed that dapsone binds to the AT-rich region of the minor groove of DNA having a relative binding energy of -6.22 kcal mol-1. Overall, all these findings evolved from this study can be used for better understanding the medicinal importance of dapsone.

Depicting the DNA binding and photo-nuclease ability of anti-mycobacterial drug rifampicin: A biophysical and molecular docking perspective.

Rifampicin, an important member of ansamycin family, exhibits various biological activities. It is frequently used for the treatment of tuberculosis and leprosy. Recently, its interaction with protein is evidenced. But, its interaction with DNA is still unknown. Whether, exhibition of anti-cancer activity of rifampicin is associated with DNA-cleavage activity is also unknown. In this study, an attempt has been taken to understand these two unknown aspects. Spectroscopic studies indicated that rifampicin binds to CT-DNA with a binding constant of ~5.22 × 105 M-1. Several independent experiments like CD analysis, competitive displacement experiments and viscosity measurements revealed that rifampicin intercalates into the CT-DNA. Molecular docking studies corroborate this fact and depicted that this drug binds to the GC-rich region of DNA through multiple hydrogen bonding having the relative binding energy of -9.21 kcal mol-1. Besides, DNA binding ability, rifampicin causes the photo-cleavage of pUC19 DNA via singlet oxygen pathway. To the best of our knowledge, we report for the first time the DNA binding and DNA cleavage ability of rifampicin. This study provides a clue behind the execution of the anti-cancer activity of rifampicin. Overall, all these information can be used for further understanding the pharmacological effects of rifampicin.

Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation.

Mycobacterium leprae, the causal agent of leprosy is non-cultivable in vitro. Thus, the assessment of antibiotic activity against Mycobacterium leprae depends primarily upon the time-consuming mouse footpad system. The GyrA protein of Mycobacterium leprae is the target of the antimycobacterial drug, Ofloxacin. In recent times, the GyrA mutation (A91V) has been found to be resistant to Ofloxacin. This phenomenon has necessitated the development of new, long-acting antimycobacterial compounds. The underlying mechanism of drug resistance is not completely known. Currently, experimentally crystallized GyrA-DNA-OFLX models are not available for highlighting the binding and mechanism of Ofloxacin resistance. Hence, we employed computational approaches to characterize the Ofloxacin interaction with both the native and mutant forms of GyrA complexed with DNA. Binding energy measurements obtained from molecular docking studies highlights hydrogen bond-mediated efficient binding of Ofloxacin to Asp47 in the native GyrA-DNA complex in comparison with that of the mutant GyrA-DNA complex. Further, molecular dynamics studies highlighted the stable binding of Ofloxacin with native GyrA-DNA complex than with the mutant GyrA-DNA complex. This mechanism provided a plausible reason for the reported, reduced effect of Ofloxacin to control leprosy in individuals with the A91V mutation. Our report is the first of its kind wherein the basis for the Ofloxacin drug resistance mechanism has been explored with the help of ternary Mycobacterium leprae complex, GyrA-DNA-OFLX. These structural insights will provide useful information for designing new drugs to target the Ofloxacin-resistant DNA gyrase.

Double differential cross sections for proton induced electron emission from molecular analogues of DNA constituents for energies in the Bragg peak region.

For track structure simulations in the Bragg peak region, measured electron emission cross sections of DNA constituents are required as input for developing parameterized model functions representing the scattering probabilities. In the present work, double differential cross sections were measured for the electron emission from vapor-phase pyrimidine, tetrahydrofuran, and trimethyl phosphate that are structural analogues to the base, the sugar, and the phosphate residue of the DNA, respectively. The range of proton energies was from 75 keV to 135 keV, the angles ranged from 15° to 135°, and the electron energies were measured from 10 eV to 200 eV. Single differential and total electron emission cross sections are derived by integration over angle and electron energy and compared to the semi-empirical Hansen-Kocbach-Stolterfoht (HKS) model and a quantum mechanical calculation employing the first Born approximation with corrected boundary conditions (CB1). The CB1 provides the best prediction of double and single differential cross section, while total cross sections can be fitted with semi-empirical models. The cross sections of the three samples are proportional to their total number of valence electrons.

TNF -308G>A single nucleotide polymorphism is associated with leprosy among Brazilians: a genetic epidemiology assessment, meta-analysis, and functional study.

Leprosy is an infectious disease caused by Mycobacterium leprae. Tumor necrosis factor (TNF) plays a key role in the host response. Some association studies have implicated the single nucleotide polymorphism TNF -308G>A in leprosy susceptibility, but these results are still controversial. We first conducted 4 association studies (2639 individuals) that showed a protective effect of the -308A allele (odds ratio [OR] = 0.77; P = .005). Next, results of a meta-analysis reinforced this association after inclusion of our new data (OR = 0.74; P = .04). Furthermore, a subgroup analysis including only Brazilian studies suggested that the association is specific to this population (OR = 0.63; P = .005). Finally, functional analyses using whole blood cultures showed that patients carrying the -308A allele produced higher TNF levels after lipopolysaccharide (LPS) (6 hours) and M. leprae (3 hours) stimulation. These results reinforce the association between TNF and leprosy and suggest the -308A allele as a marker of disease resistance, especially among Brazilians.

Production of volatile organic compounds (VOCs) by yeasts isolated from the ascocarps of black (Tuber melanosporum Vitt.) and white (Tuber magnatum Pico) truffles.

Twenty-nine yeast strains were isolated from the ascocarps of black and white truffles (Tuber melanosporum Vitt. and Tuber magnatum Pico, respectively), and identified using a polyphasic approach. According to the conventional taxonomic methods, MSP-PCR fingerprinting and sequencing of the D1/D2 domain of 26S rDNA, the strains were identified as Candida saitoana, Debaryomyces hansenii, Cryptococcus sp., Rhodotorula mucilaginosa, and Trichosporon moniliiforme. All isolates assimilated L: -methionine as a sole nitrogen source and produced the volatile organic compounds (VOCs), 2-methyl butanol, 3-methyl butanol, methanethiol, S-methyl thioacetate, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, dihydro-2-methyl-3(2H)-thiophenone and 3-(methylthio)-1-propanol (MTP). ANOVA analysis of data showed significant (P<0.01) differences in VOCs produced by different yeasts, with MTP as the major component (produced at concentrations ranging from 19.8 to 225.6 mg/l). In addition, since some molecules produced by the isolates of this study are also characteristic of truffle complex aroma, it is possible to hypothesize a complementary role of yeasts associated with this ecosystem in contributing to final Tuber spp. aroma through the independent synthesis of yeast-specific volatile constituents.

Single nucleotide polymorphisms (SNPs) at -238 and -308 positions in the TNFalpha promoter: clinical and bacteriological evaluation in leprosy.

Tumor necrosis factor alpha (TNFalpha) plays a key role in orchestrating the complex events involved in inflammation and immune response. The presence of single nucleotide polymorphisms (SNPs) within the promoter region of the TNFa gene has been associated with a number of diseases. The aim of this study was to investigate the distribution of polymorphisms at positions -238 (G/A) and -308 (G/A) at the TNFalpha promoter, and its association to the outcome of different clinical forms of leprosy. Furthermore, the bacteriological index (BI) was evaluated among genotyped multibacillary (MB) patients in order to investigate the possible influence of each polymorphism on the bacterial load. This study included a total of 631 leprosy patients being 401 MB and 230 paucibacillary (PB), that was further separated according to its ethnicity (Afro- and Euro-Brazilians). The combination of SNPs in haplotypes generated three different arrangements: TNFG-G, TNFG-A and TNFA-G. In spite of the marked differences observed in the frequency of the haplotypes along the ethnic groups, no statistical differences were observed in haplotype frequencies between MB and PB patients. The BI analyses showed a lower bacteriological index among the -308 carriers, while the BI of the -238 carriers was higher. Although no significance has been achieved in this analysis regarding the influence of the polymorphisms to the development of the clinical outcome, it seems that in a different stage (among the MB patients) the polymorphisms could contribute to the degree of severity observed.

Functional interactions of Mycobacterium leprae RuvA with Escherichia coli RuvB and RuvC on holliday junctions.

The Mycobacterium leprae RuvA homologue (MlRuvA) was over-expressed in Escherichia coli and purified to homogeneity. The DNA-binding specificity and the functional interactions of MlRuvA with E. coli RuvB and RuvC (EcRuvB and EcRuvC) were examined using synthetic Holliday junctions. MlRuvA bound specifically to Holliday junctions and produced similar band-shift patterns as EcRuvA. Moreover, MlRuvA formed functional DNA helicase and branch-migration enzymes with EcRuvB, although the heterologous enzyme had a lower efficiency. These results demonstrate that the RuvA homologue of M. leprae is a functional branch-migration subunit. Whereas MlRuvA promoted branch-migration in combination with EcRuvB, it was unable to stimulate branch-migration-dependent resolution in a RuvABC complex. The inability to stimulate RuvC was not due to its failure to form heterologous RuvABC complexes on junctions, since such complexes were detected by co-immunoprecipitation. Most likely, the stability of the heterologous RuvABC complex and, possibly, the interactions between RuvA and RuvC were impaired, as gel-shift experiments failed to show mixed MlRuvA-EcRuvC-junction complexes. These results demonstrate that branch-migration per se and the assembly of a RuvABC complex on the Holliday junction are insufficient for RuvAB-dependent resolution of the junction by RuvC, suggesting that specific and intimate interactions between all three proteins are required for the function of a RuvABC "resolvasome".

Comparison of the T cell patterns in leprous and cutaneous sarcoid granulomas. Presence of Valpha24-invariant natural killer T cells in T-cell-reactive leprosy together with a highly biased T cell receptor Valpha repertoire.

The T-cell-reactive (eg, tuberculoid and reversal) forms of leprosy represent a well-defined granulomatous reaction pattern against an invading pathogen. The immune response in cutaneous sarcoidosis is a granulomatous condition that pathologically is very similar to T-cell reactive leprosy. However, it lacks a defined causative agent. In view of the role of NKT cells in murine granulomas induced by mycobacterial cell walls, we have searched for the presence of NKT cells in the cutaneous lesions of both leprosy and sarcoidosis. These cells were present in T-cell-reactive leprosy but were undetectable in cutaneous sarcoidosis. We have also studied the TCR Valpha repertoire in the two diseases. In addition to Valpha24(+) NKT cells, all patients with T-cell-reactive leprosy showed a very restricted T-cell-reactive Valpha repertoire with a strong bias toward the use of the Valpha6 and Valpha14 segments. Valpha6 and Valpha14(+) T cells were polyclonal in terms of CDR3 length and Jalpha usage. In contrast, most sarcoidosis patients showed a diverse usage of Valpha chains associated with clonal or oligoclonal expansions reminiscent of antigen-driven activation of conventional T cells. Thus the origin and perpetuation of the two kinds of granulomatous lesions appear to depend on altogether distinct T-cell recruiting mechanisms.

A synthetic holliday junction is sandwiched between two tetrameric Mycobacterium leprae RuvA structures in solution: new insights from neutron scattering contrast variation and modelling.

The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Holliday junction. To study the solution structure of this complex, contrast variation by neutron scattering was applied to Mycobacterium leprae RuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA junction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their complex, respectively, showing that the complex was structurally more extended than MleRuvA. No difference was observed in the presence or absence of Mg2+. The large difference in RG values for the free and complexed protein in 65% 2H2O, where the DNA component is "invisible", showed that a substantial structural change had occurred in complexed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the complex were 19 and 15 or less (x10(-5)), respectively, indicating that DNA passed through the centre of the complex. Automated constrained molecular modelling based on the Escherichia coli RuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% 2H2O is explained by a face-to-face association of two MleRuvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% 2H2O showed that the two tetramers are separated by a void space of about 1-2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and complexed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought.

Spectroscopic investigation of Tet repressor tryptophan-43 upon specific and nonspecific DNA binding.

The F75 Tet repressor mutant (F75 TetR) contains a single tryptophan residue located at position 43 in the operator recognition alpha-helix. Previous studies [Hansen, D., & Hillen, W. (1987) J. Biol. Chem. 262, 12269-12274] have shown that the fluorescence intensity of this residue is dramatically reduced upon operator binding. In order to determine the origin of this quenching and the role of Trp-43 in the binding mechanism, we have investigated its fluorescence properties upon F75 TetR binding to a tet operator containing 76 bp DNA fragment (specific binding) and to sheared calf thymus DNA (nonspecific binding). Trp-43 steady-state fluorescence intensity was quenched by 72% upon specific binding and by 45% upon nonspecific binding. These fluorescence intensity decreases were not accounted for by similar decreases in the respective fluorescence lifetimes. The apparent quenching calculated from the average lifetimes was about 0.33 in both binding modes. This shows the presence of a static quenching process, clearly favored upon specific binding as compared to nonspecific binding. This is consistent with stacking interactions between Trp-43 and the DNA bases, as suggested by molecular graphics [Baumeister, R., Helbl, V., & Hillen, W. (1992) J. Mol. Biol. 226, 1257-1270]. The equilibrium constant between nonfluorescent and fluorescent tryptophan residues was 5 times higher upon binding to specific DNA than to nonspecific DNA. The preferential static quenching of Trp-43 in the specific complex suggests that stacking interactions might contribute to the specific binding mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of chromatin folding by histone acetylation.

A homogeneous oligonucleosome complex was prepared by reconstitution of highly hyperacetylated histone octamers onto a linear DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Lytechinus variegatus. The ionic strength-dependent folding of this oligonucleosome assembly was monitored by sedimentation velocity and electron microscopy. Both types of analysis indicate that under ionic conditions resembling those found in the physiological range and in the absence of histone H1, the acetylated oligonucleosome complexes remain in an extended conformation in contrast to their nonacetylated counterparts. The implications of this finding in the context of a multistate model of chromatin folding (Hansen, J. C., and Ausio, J. (1992) TIBS 197, 187-191) as well as its biological relevance are discussed.