Combinatorial identification of a novel consensus sequence for the covalent DNA-binding polyamide tallimustine

Many agents successfully used in cancer chemotherapy either directly or indirectly covalently modify DNA. Examples include cisplatin, which forms a covalent adduct with guanines, and doxorubicin, which traps a cleavage intermediate between topoisomerase II and torsionally strained DNA. In most cases, the efficacy of these drugs depends on the efficiency and specificity of their DNA binding, as well as the discrimination between normal and neoplastic cells in their handling of the drug-DNA adducts. While much is known about the chemistry of drug-DNA adducts, little is known regarding the overall specificity of their formation, especially in the context of a whole human genome, where potentially billions of binding sites are possible. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA-binding specificity of the semisynthetic covalent DNA-binding polyamide tallimustine, which contains a benzoic acid nitrogen mustard appended to the minor groove DNA-binding natural product distamycin A. After investigating over 134 million possible sequences, we found that the highest affinity tallimustine binding sites contained one of two consensus sequences, either the expected distamycin hexamer binding sites followed by a CG base pair (e.g., 5'-TTTTTTC-3' and 5'-AAATTTC-3') or the unexpected sequence 5'-TAGAAC-3'. Curiously, we found that tallimustine preferentially alkylated the N7 position of guanines located on the periphery of these consensus sequences. These findings suggested a cooperative binding model for tallimustine in which one molecule noncovalently resides in the DNA minor groove and locally perturbs the DNA structure, thereby facilitating alkylation by a second tallimustine of an exposed guanine on another side of the DNA.     

Design, synthesis, and evaluation of novelly substituted benzimidazole compounds as angiotensin II receptor antagonists

5-Nitrobenzimidazole derivatives with varying substituents at 2-position have been designed, synthesized, and evaluated for angiotensin II antagonistic activity. A drug-receptor interaction model has been proposed.     

Compartment-specific Synthesis of Phosphatidylethanolamine Is Required for Normal Heavy Metal Resistance

Control of lipid composition of membranes is crucial to ensure normal cellular functions. Saccharomyces cerevisiae has two different phosphatidylserine decarboxylase enzymes (Psd1 and Psd2) that catalyze formation of phosphatidylethanolamine. The mitochondrial Psd1 provides roughly 70% of the phosphatidylethanolamine (PE) biosynthesis in the cell with Psd2 carrying out the remainder. Here, we demonstrate that loss of Psd2 causes cells to acquire sensitivity to cadmium even though Psd1 remains intact. This cadmium sensitivity results from loss of normal activity of a vacuolar ATP-binding cassette transporter protein called Ycf1. Measurement of phospholipid levels indicates that loss of Psd2 causes a specific reduction in vacuolar membrane PE levels, whereas total PE levels are not significantly affected. The presence of a phosphatidylinositol transfer protein called Pdr17 is required for Psd2 function and normal cadmium tolerance. We demonstrate that Pdr17 and Psd2 form a complex in vivo that seems essential for maintenance of vacuolar PE levels. Finally, we refine the localization of Psd2 to the endosome arguing that this enzyme controls vacuolar membrane phospholipid content by regulating phospholipids in compartments that will eventually give rise to the vacuole. Disturbance of this regulation of intracellular phospholipid balance leads to selective loss of membrane protein function in the vacuole.     

Oxygen Therapy Use in Older Adults with Chronic Obstructive Pulmonary Disease

Rationale

Oxygen therapy improves survival and function in severely hypoxemic chronic obstructive pulmonary disease (COPD) patients based on two landmark studies conducted over 40 years ago. We hypothesize that oxygen users in the current era may be very different. We examined trends and subject characteristics associated with oxygen therapy use from 2001–2010 in the United States.

Methods

We examined Medicare beneficiaries with COPD who received oxygen from 2001 to 2010. COPD subjects were identified by: 1) ≥2 outpatient visits >30 days apart within one year with an encounter diagnosis of COPD; or 2) an acute care hospitalization with COPD as the primary or secondary discharge diagnosis. Oxygen therapy and sustained oxygen therapy were defined as ≥1 and ≥11 claims for oxygen, respectively, in the durable medical equipment file in a calendar year. Primary outcome measures were factors associated with oxygen therapy and sustained oxygen therapy over the study period.

Results

Oxygen therapy increased from 33.7% in 2001 to 40.5% in 2010 (p-value of trend <0.001). Sustained oxygen therapy use increased from 19.5% in 2001, peaked in 2008 to 26.9% and declined to 18.5% in 2010. The majority of subjects receiving oxygen therapy and sustained oxygen therapy were female. Besides gender, factors associated with any oxygen use or sustained oxygen therapy were non-Hispanic white race, low socioeconomic status and ≥2 comorbidities.

Conclusions

Any oxygen use among fee-for service Medicare beneficiaries with COPD is high. Current users of oxygen are older females with multiple comorbidities. Decline in sustained oxygen therapy use after 2008 may be related to reimbursement policy change.     

Interaction pattern for the complex of B-DNA Fullerene compounds with a set of known replication proteins using docking study

Fullerenes have attracted considerable attention due to their unique chemical structure and potential applications which has opened wide venues for possible human exposure to various fullerene types. Therefore, in depth knowledge of how fullerene may interfere with various cellular processes becomes quite imperative. The present study was designed to investigate how the presence of fullerene affect the binding of DNA with different enzymes involved in replication process. Different fullerenes were first docked with DNA and then binding scores of different enzymes was analyzed with fullerene docked DNA. C30, C40 & C50 once docked with DNA, reduced the binding score of primase, whereas no significant change in the binding score was observed with the helicase, ssb protein, dna pol δ, dna pol ε, ligase, DNA clamp, and topoisomerases. On the contrast, the binding score of RPA14 decreases in fluctuating manner while interacting with increasing molecular weight of fullerene bound single-stranded DNA complex. The study revealed the affect of fullerene family interacting with DNA on the binding pattern of enzymes involved in replication process. Study suggests that the presence of most of fullerenes may not affect the activity of these enzymes necessary for replication process whereas C30, C40 & C50 may disrupt the activity of primase, (strating point for DNA polymerase) its docking score decreases from 13820 to 10702.     

Antiviral potential of 4-hydroxypanduratin A,secondary metabolite of Fingerroot,Boesenbergia pandurata (Schult.), towards Japanese Encephalitis virus NS2B/NS3 protease

4-hydroxypanduratin A is a secondary metabolite of Boesenbergia pandurata Schult. (Fingerroot) plant with various pharmacological activities such as neuroprotective, potent antioxidant, antibacterial and antifungal. Flaviviral NS2B/NS3 protease activity is essential for polyprotein processing and viral replication for Japanese Encephalitis Virus (JEV), a major cause of Acute Encephaltis in Asia. Inhibition of formation of this complex by arresting the binding of NS2B with NS3 would reduce the enzyme''s activity to meager proportions and hence would prevent further viral proliferation. The automated 3D structure of NS2B protein of the JEV GP78 was predicted based on the sequence-to-structure-to-function paradigm using I-TASSER and the function of NS2B protein was inferred by matching to other known proteins. The stereochemical quality of predicted structure was checked by PROCHECK. The antiviral activity of 4-hydroxypanduratin A against NS2B protein as a potential drug has been elucidated in this paper. Docking simulation analysis showed 4-hydroxypanduratin A as potential inhibitor of NS2B protein/cofactor which is necessary for NS3 protease activity. 220 derivatives of 4-hydroxypanduratin A were virtually screened with rigid criteria of Lipinski''s rule of 5 using Autodock4.2. 4-hydroxypanduratin A was found interacting with target hydrophilic domain in NS2B protein by two Hbonds (Gly80 and Asp81) with active residues, several hydrophobic interactions, Log P value of 5.6, inhibition constant (Ki) of 51.07nM and lowest binding energy of -9.95Kcal/Mol. Hence, 4-hydroxypanduratin A targeted to Site 2 will have sufficient profound effect to inhibit protease activity to abrogate viral replication. It could be a promising potential drug candidate for JEV infections using NS2B Site 2 as a Drug target.     

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses ‘nodular calli’ developed on the BAP - pretreated daughter frond explants in B₅ medium containing sucrose (1.0 %) with 2,4-D (5.0 μM) and 2-iP (50.0 μM) or 2,4-D (50.0 μM) and TDZ (5.0 μM) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for β-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11–13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.