Molecular basis of recognition of quadruplexes human telomere and c-myc promoter by the putative anticancer agent sanguinarine

Background

Interaction of putative anticancer agent sanguinarine with two quadruplex forming sequences, human telomeric DNA (H24) and NHE III₁ upstream of the P1 promoter of c-myc (Pu27), has been studied to understand the structural basis of the recognition.

Methods

Absorption, fluorescence and circular dichroism spectroscopy have been employed to characterize the association. Energetics of the interaction was studied by isothermal titration and differential scanning calorimetry. TRAP assay was done to assess the inhibitory potential of sanguinarine.

Results

Absorption and fluorescence studies show that sanguinarine has high binding affinity of ~ 10⁵ M^− 1 for both sequences. Binding stoichiometry is 2:1 for H24 and 3:1 for Pu27. Results suggest stacking interaction between planar sanguinarine moiety and G-quartets. Circular dichroism spectra show that sanguinarine does not cause structural perturbation in the all-parallel Pu27 but causes a structural transition from mixed hybrid to basket form at higher sanguinarine concentration in case of H24. The interaction is characterized by total enthalpy–entropy compensation and high heat capacity values. Differential scanning calorimetry studies suggest that sanguinarine binding increases the melting temperature and also the total enthalpy of transition of both quadruplexes. TRAP results show that sanguinarine effectively blocks telomerase activity in a concentration dependent manner in cell extracts from MDAMB-231 breast cancer cell lines.

Conclusion

These results suggest that there is a difference in the structural modes of association of sanguinarine to the quadruplexes.

General significance

It helps to understand the role of quadruplex structures as a target of small molecule inhibitors of telomerase.     

Genome-wide study predicts promoter-G4 DNA motifs regulate selective functions in bacteria: radioresistance of D. radiodurans involves G4 DNA-mediated regulation

A remarkable number of guanine-rich sequences with potential to adopt non-canonical secondary structures called G-quadruplexes (or G4 DNA) are found within gene promoters. Despite growing interest, regulatory role of quadruplex DNA motifs in intrinsic cellular function remains poorly understood. Herein, we asked whether occurrence of potential G4 (PG4) DNA in promoters is associated with specific function(s) in bacteria. Using a normalized promoter-PG4-content (PG4_P) index we analysed >60 000 promoters in 19 well-annotated species for (a) function class(es) and (b) gene(s) with enriched PG4_P. Unexpectedly, PG4-associated functional classes were organism specific, suggesting that PG4 motifs may impart specific function to organisms. As a case study, we analysed radioresistance. Interestingly, unsupervised clustering using PG4_P of 21 genes, crucial for radioresistance, grouped three radioresistant microorganisms including Deinococcus radiodurans. Based on these predictions we tested and found that in presence of nanomolar amounts of the intracellular quadruplex-binding ligand N-methyl mesoporphyrin (NMM), radioresistance of D. radiodurans was attenuated by ∼60%. In addition, important components of the RecF recombinational repair pathway recA, recF, recO, recR and recQ genes were found to harbour promoter-PG4 motifs and were also down-regulated in presence of NMM. Together these results provide first evidence that radioresistance may involve G4 DNA-mediated regulation and support the rationale that promoter-PG4s influence selective functions.     

Pathogen virulence of Phytophthora infestans: from gene to functional genomics

The oomycete, Phytophthora infestans, is one of the most important plant pathogens worldwide. Much of the pathogenic success of P. infestans, the potato late blight agent, relies on its ability to generate large amounts of sporangia from mycelia, which release zoospores that encyst and form infection structures. Until recently, little was known about the molecular basis of oomycete pathogenicity by the avirulence molecules that are perceived by host defenses. To understand the molecular mechanisms interplay in the pathogen and host interactions, knowledge of the genome structure was most important, which is available now after genome sequencing. The mechanism of biotrophic interaction between potato and P. infestans could be determined by understanding the effector biology of the pathogen, which is until now poorly understood. The recent availability of oomycete genome will help in understanding of the signal transduction pathways followed by apoplastic and cytoplasmic effectors for translocation into host cell. Finally based on genomics, novel strategies could be developed for effective management of the crop losses due to the late blight disease.     

Role of heavy metal resistant Ochrobactrum sp. and Bacillus spp. strains in bioremediation of a rice cultivar and their PGPR like activities

The present study demonstrates the metal toxicity ameliorating and growth promoting abilities of three different bacterial isolates when applied to rice as host plant. The three bacterial strains included a cadmium resistant Ochrobactrum sp., a lead resistant Bacillus sp. and an arsenic resistant Bacillus sp. designated as CdSP9, PbSP6, and AsSP9, respectively. When these isolates were used as inocula applied to metal-treated rice plants of variety Satabdi, the germination percentage, relative root elongation (RRE), amylase and protease activities were increased. The toxic effect of metal was reduced in presence of these bacteria. The overall biomass and root/shoot ratio were also enhanced by bacterial inoculation. Hydroponic studies showed that the superoxide dismutase (SOD) activity and malondialdehyde (MDA) level, which had been increased in the presence of metal stress in rice roots, were lowered by the bacterial inoculation. In addition, all three strains were 1-aminocyclopropane-1-carboxylate (ACC) deaminase and catalase positive, whereas siderophore producing ability was lacking in PbSP6. However, both PbSP6 and AsSP9 were protease positive and could hydrolyse starch. The data indicate that these bacteria have promise for bioremediation as well as for plant growth promotion.     

An audit strategy for progression-free survival

In randomized clinical trials, the use of potentially subjective endpoints has led to frequent use of blinded independent central review (BICR) and event adjudication committees to reduce possible bias in treatment effect estimators based on local evaluations (LE). In oncology trials, progression-free survival (PFS) is one such endpoint. PFS requires image interpretation to determine whether a patient's cancer has progressed, and BICR has been advocated to reduce the potential for endpoints to be biased by knowledge of treatment assignment. There is current debate, however, about the value of such reviews with time-to-event outcomes such as PFS. We propose a BICR audit strategy as an alternative to a complete-case BICR to provide assurance of the presence of a treatment effect. We develop an auxiliary-variable estimator of the log-hazard ratio that is more efficient than simply using the audited (i.e., sampled) BICR data for estimation. Our estimator incorporates information from the LE on all the cases and the audited BICR cases, and is an asymptotically unbiased estimator of the log-hazard ratio from BICR. The estimator offers considerable efficiency gains that improve as the correlation between LE and BICR increases. A two-stage auditing strategy is also proposed and evaluated through simulation studies. The method is applied retrospectively to a large oncology trial that had a complete-case BICR, showing the potential for efficiency improvements.     

Immunomodulatory effect of picroliv on the efficacy of paromomycin and miltefosine in combination in experimental visceral leishmaniasis

Combination therapy for the treatment of visceral leishmaniasis has increasingly been advocated as a way to increase treatment efficacy and tolerance, to reduce treatment duration and cost, and to limit the emergence of drug resistance. In the present work, we have adopted a rational approach, which can modulate the immune response to overcome the negative control systems and to boost the positive killing responses. This study was designed to investigate the immunomodulatory effect of picroliv (standardized fraction from the alcoholic extract of root and rhizome of Picrorhiza kurroa) on a combination of paromomycin and miltefosine using Leishmania donovani/hamster model. Picroliv has significantly enhanced antileishmanial efficacy and lymphocyte proliferation when given in combination with paromomycin and miltefosine. Increased toxic oxygen metabolite generation and phagocytosis were also witnessed. Present study thus establishes the possible use of picroliv as adjunct to antileishmanial chemotherapy.     

Increased enzyme secretion by 2-deoxy-D-glucose in presence of succinate by suppression of metabolic enzymes in Termitomyces clypeatus

Regulatory mode of secretion of proteins was detected for the industrial glycosidase, cellobiase, under secreting conditions (in presence of TCA cycle intermediates like succinate etc.) in the filamentous fungus Termitomyces clypeatus. The titers of key metabolic enzymes were investigated under secreting and non-secreting conditions of growth and compared to the corresponding production of intra and extracellular levels of cellobiase. Results were compared in presence of 2-deoxy-D-glucose, a potent glycosylation inhibitor in the secreting media. Inclusion of 2-deoxy-D-glucose in presence of succinate caused about 10 to 100 times decrease in titers of the metabolic enzymes hexokinase, fructose-1,6-bisphosphatase, isocitrate lyase and malate dehydrogenase leading to increased secretion of cellobiase by more than 100 times. The intracellular concentration of cAMP (86-fold decrease in presence of 2-deoxy-D-glucose under secreting conditions) and turnover rate of proteins also dropped significantly. In this suppressed metabolic state, a 10-fold increase in the titer of the secreted cellobiase was noticed. The results indicated elucidation of carbon catabolite repression like phenomenon in the fungus under secreting conditions which was more pronounced by 2-deoxy-D-glucose. The interdependence between secretion and regulation of metabolic enzymes will help in better understanding of the physiology of these highly adapted organisms for increasing their secretion potential of glycosidases like cellobiase with high industrial value.     

Expedited Baeyer-Villiger oxidation of steroidal ketones by microwave irradiation

Microwave (MW) assisted reactions are currently having considerable importance in the synthesis of organic compounds. Considering the remarkable application of Baeyer–Villiger (BV) reaction in the synthesis of natural products and steroid–peptide conjugates, we report here some of our findings of BV oxidation of carbonyl compounds with special reference to steroidal ketones under MW irradiation justifying its accelerating effect.     

Distribution of Coliphages Against Four <Emphasis Type="Italic">E. Coli</Emphasis> Virotypes in Hospital Originated Sewage Sample and a Sewage Treatment Plant in Bangladesh

The distribution of coliphages infecting different Escherichia coli virotypes (EHEC, EIEC, EPEC, ETEC) and an avirulent strain (K-12) in sewage system of a hospital and a sewage treatment plant (STP) was investigated by culture-based agar overlay methods. Coliphages were found in all the samples except stool dumping site in the sewage system of the hospital and lagoon of the STP. Bacteriophage count (pfu/ml) infecting E. coli strains showed the following ascending pattern (EHEC < EIEC < EPEC < ETEC < E coli K-12) in all the collected samples except one. Phages capable of infecting avirulent E. coli K-12 strains were present in the highest number among all the examined locations. Phages specific for E. coli K-12 presented high diversity in plaque size on the bacterial lawn. Virulent E. coli specific coliphages rarely produced plaques with diameter of 1–2 mm or over. Conventional agar overlay method was found to be not satisfactory for phage community analysis from primary stool dumping site of the hospital, probably due to the presence of high concentration of antimicrobial substances. The gradual decrease seen in the five groups of coliphage quantity with the ongoing treatment process and then the absolute absence of coliphages in the outlet of the examined treatment plant is indicative of the usefulness of the treatment processes practiced there.     

Mechanism of amylin fibrillization enhancement by heparin

We characterized the interaction of amylin with heparin fragments of defined length, which model the glycosaminoglycan chains associated with amyloid deposits found in type 2 diabetes. Binding of heparin fragments to the positively charged N-terminal half of monomeric amylin depends on the concentration of negatively charged saccharides but is independent of oligosaccharide length. By contrast, amylin fibrillogenesis has a sigmoidal dependence on heparin fragment length, with an enhancement observed for oligosaccharides longer than four monomers and a leveling off of effects beyond 12 monomers. The length dependence suggests that the negatively charged helical structure of heparin electrostatically complements the positively charged surface of the fibrillar amylin cross-β structure. Fluorescence resonance energy transfer and total internal reflection fluorescence microscopy experiments indicate that heparin associates with amylin fibrils, rather than enhancing fibrillogenesis catalytically. Short heparin fragments containing two- or eight-saccharide monomers protect against amylin cytotoxicity toward a MIN6 mouse cell model of pancreatic β-cells.