The effect of oxidatively damaged DNA on the active site pre-organization during nucleotide incorporation in a high fidelity polymerase from \u3cem\u3eBacillus stearothermophilus\u3c/em\u3e

We study the effect of the oxidative lesion 8-oxoguanine (8oxoG) on the pre-organization of the active site for DNA replication in the closed (active) state of the Bacillus Fragment (BF), a Klenow analog from Bacillus stearothermophilus. Our molecular dynamics and free energy simulations of explicitly solvated model ternary complexes of BF bound to correct dCTP/incorrect dATP opposite guanine (G) and 8oxoG bases in DNA suggest that the lesion introduces structural and energetic changes at the catalytic site to favor dATP insertion. Despite the formation of a stable Watson-Crick pairing in the 8oxoG:dCTP system, the catalytic geometry is severely distorted to possibly slow down catalysis. Indeed, our calculated free energy landscapes associated with active site pre-organization suggest additional barriers to assemble an efficient catalytic site, which need to be overcome during dCTP incorporation opposite 8oxoG relative to that opposite undamaged G. In contrast, the catalytic geometry for the Hoogsteen pairing in the 8oxoG:dATP system is highly organized and poised for efficient nucleotide incorporation via the twometal- ion catalyzed phosphoryl transfer mechanism. However, the free energy calculations suggest that the catalytic geometry during dATP incorporation opposite 8oxoG is considerably less plastic than that during dCTP incorporation opposite G despite a very similar, well organized catalytic site for both systems. A correlation analysis of the dynamics trajectories suggests the presence of significant coupling between motions of the polymerase fingers and the primary distance for nucleophilic attack (i.e., between the terminal primer O3´ and the dNTP Pα atoms) during correct dCTP incorporation opposite undamaged G. This coupling is shown to be disrupted during nucleotide incorporation by the polymerase with oxidatively damaged DNA/dNTP substrates. We also suggest that the lesion affects DNA interactions with key polymerase residues, thereby affecting the enzymes ability to discriminate against noncomplementary DNA/dNTP substrates. Taken together, our results provide a unified structural, energetic, and dynamic platform to rationalize experimentally observed relative nucleotide incorporation rates for correct dCTP/incorrect dATP insertion opposite an undamaged/oxidatively damaged template G by BF

Computational Study of the Force Dependence of Phosphoryl Transfer during DNA Synthesis by a High Fidelity Polymerase

High fidelity polymerases are efficient catalysts of phosphodiester bond formation during DNA replication or repair. We interpret molecular dynamics simulations of a polymerase bound to its substrate DNA and incoming nucleotide using a quasiharmonic model to study the effect of external forces applied to the bound DNA on the kinetics of phosphoryl transfer. The origin of the force dependence is shown to be an intriguing coupling between slow, delocalized polymerase-DNA modes and fast catalytic site motions. Using noncognate DNA substrates we show that the force dependence is context specific

Preparation and characterization of protein-nanotube conjugates

This chapter describes methods of immobilizing proteins on carbon nanotubes, using two different routes—physical adsorption and covalent attachment. We also provide an overview on how such conjugates can be characterized with the help of various techniques, such as Raman, Fourier transform infrared (FT-IR), circular dichroism (CD), and fluorescence spectroscopies, in addition to the standard enzyme kinetic analyses of activity and stability. Both the attachment routes—covalent and noncovalent—could be used to prepare protein conjugates that retained a significant fraction of their native structure and function; furthermore, the protein conjugates were operationally stable, reusable, and functional even under harsh denaturing conditions. These studies therefore corroborate the use of these immobilization methods to engineer functional carbon nanotube-protein hybrids that are highly active and stable

Clinical manifestation and prevalence of peripheral neuropathy and nerve dysfunction in patients with chronic kidney disease

Background: Chronic Kidney Disease (CKD) is recognized as a major health problem. Prevalence of CKD is rising continuously; mostly CKD is affecting the elderly aged population and/or patients with diabetes and hypertension. Present study was aimed to explore clinical manifestation and evaluate the prevalence of peripheral neuropathy and peripheral nerve dysfunction in CKD patients attending our hospital with reference to the severity and duration of the CKD.Methods: The present cross sectional study was conducted in 74 patients affected with chronic kidney disease, of different age groups at the medical wards of King George Hospital, Visakhapatnam. The presence of peripheral nerve dysfunction was assessed by nerve dysfunction clinically (motor or sensory symptoms and signs) and electrophysiological nerve conduction studies.Results: Out of 74 patients, 65% of study population was suffering from chronic kidney disease with peripheral nerve dysfunction. The peripheral nerves dysfunction was more prevalent in elder age (>65 years) subjects when compared to subjects with age <65 years. Moreover, the results shown that the rate of prevalence of peripheral nerves dysfunction was observed higher in subjects with longer duration of CKD. Male subjects were affected more when creatinine clearance is <15 ml/minute. Both sexes were affected equally when creatinine clearance is between 30-59 ml/minute.Conclusion: This study enlightens the prevalence and clinical presentation of peripheral nerve dysfunction in patients with CKD. The CKD was found to cause peripheral neuropathy including overt and subclinical neuropathy, of which distal symmetrical sensory motor neuropathy was common in CKD. The prevalence of peripheral neuropathy was directly proportional to duration and severity of CKD.

A Validated Ion Chromatography method for determination of Ammonium content in Omeprazole tablets

A simple, feasible, definite and strong Ion chromatography method was developed for the quantitative determination of ammonium content in Omeprazole tablets. The method was developed using Ion pac CS17 Column, 250 X 4.6mm X 5.0 m column with mobile phase containing 1.5mM Methane sulfonic acid in water. The eluted compounds were monitored using conductivity detector. The unknown peak and ammonium peak were well separated with resolution more than 2.0. The developed method was validated as per Internatonal Conference on Harmonisation of technical Requirements for registration of pharmaceuticals for human use guidelines with respect to linearity (The high correlation coefficient >0.99), limit of detection, limit of quantification, exactness, precision and robustness. The Limit of detection, Limit of quantification values of Ammonium were 8ppm and 30ppm respectively

EVALUATION OF IN VITRO ANTI-INFLAMMATORY ACTIVITY AND HPTLC ANALYSIS OF PLANT PHYLLANTHUS FRATERNUS

Objective: The present investigation evaluated in vitro anti-inflammatory activity of Phyllanthus fraternus. Inhibition of Cyclooxygenase and 5-lipoxygenase was performed along with protein denaturation.Methods: Alcoholic extract of plant was subjected to in vitro anti-inflammatory activity and HPTLC analysis.Results: The results of anti-inflammatory activity showed significant inhibition in Cyclooxygenase and 5-lipoxygenase assay, extract also showed more than 70 % inhibition in protein denaturation method. HPTLC of plant materials was also performed; spots of alkaloids were recorded.Conclusion: Different alkaloids were spotted in chromatographic analysis and study suggested that anti-inflammatory activity of Phyllanthus fraternus may be due to the presence of alkaloids.Â

Phytochemical and Anti-Inflammatory Evaluation of Herbal Gel Prepared from Bark Extract of Mesua Ferrea Linn

The present research work involves formulation of herbal gel containing stem bark extracts of Mesua ferrea Linn and its evaluation for in vitro anti-inflammatory activity. The gel formulations were prepared using ethanolic extracts along with different polymer. The physiochemical parameters of formulations like; pH, viscosity and spreadability etc. also evaluated. Phytochemical analysis revealed presence of phenols, flavonoids and alkaloids, etc. The ethanolic extracts showed appreciable anti-inflammatory activity compared to the standard drug. Study confirmed that potential anti-inflammatory formulation can be developed from bark extract of Mesua ferrea Linn.&nbsp;&nbsp; Keywords: Mesua ferrea Linn., Anti-inflammatory, Herbal Gel, Phytochemical, Albumin denaturation