Showing papers by "Deepak Sharma published in 2011"

Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p.

Abstract: Organisms encode multiple homologous heat shock protein (Hsp)-70s, which are essential protein chaperones that play the major role in cellular protein “quality control.” Although Hsp70s are functionally redundant and highly homologous, many possess distinct functions. A regulatory motif underlying such distinctions, however, is unknown. The 98% identical cytoplasmic Hsp70s Ssa1p and Ssa2p function differently with regard to propagation of yeast [URE3] prions and in the vacuolar-mediated degradation of gluconeogenesis enzymes, such as FBPase. Here, we show that the Hsp70 nucleotide binding domain (NBD) regulates these functional specificities. We find little difference in ATPase, protein refolding, and amyloid inhibiting activities of purified Ssa1p and Ssa2p, but show that interchanging NBD residue alanine 83 (Ssa1p) and glycine 83 (Ssa2p) switched functions of Ssa1p and Ssa2p in [URE3] propagation and FBPase degradation. Disrupting the degradation pathway did not affect prion propagation, however, indicating these are two distinct processes where Ssa1/2p chaperones function differently. Our results suggest that the primary evolutionary pressure for Hsp70 functional distinctions is not to specify interactions of Hsp70 with substrate, but to specify the regulation of this activity. Our data suggest a rationale for maintaining multiple Hsp70s and suggest that subtle differences among Hsp70s evolved to provide functional specificity without affecting overall enzymatic activity.

Simultaneous estimation of amlodipine besylate and nebivolol hydrochloride in tablet dosage forms by reverse phase-high-performance liquid chromatographic using ultraviolet detection.

Abstract: Background: The present study aimed to develop and validate the simultaneous estimation of amlodipine and nebivolol in tablet dosage forms. Materials and Methods: An isocratic reversed phase high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 268 nm has been developed for the determination of amlodipine besylate (ADB) and nebivolol hydrochloride in dosage formulation. Results: Good chromatographic separation was achieved by using a stainless steel analytical column, the Lichrospher ODS RP-18 column (250 × 4 mm), particle size 5 μm. The system was operated at ambient temperature (25 ± 2°C) using a mobile phase consisting of acetonitrile (ACN) and a phosphate buffer (pH 3.0), mixed in a ratio of 40 : 60 at a flow rate of 0.8 ml/minute. The slope, intercept, and correlation coefficient were found to be 8818.2, - 18159, and 0.9993 for amlodipine and 9048.7, 108595, and 0.9998 for nebivolol, respectively. The proposed method was validated for its specificity, linearity, accuracy, and precision. Conclusion: The method was found to be suitable for the quality control of amlodipine besylate and nebivolol hydrochloride simultaneously in a bulk drug as well as in a formulation. Key words: Amlodipine besylate, isocratic separation, method validation, nebivolol hydrochloride, RP-HPLC.

Mapping mouse IL‐13 binding regions using structure modeling, molecular docking, and high‐density peptide microarray analysis

Abstract: Interleukin-13 is a Th2-associated cytokine responsible for many pathological responses in allergic asthma including mucus production, inflammation and extracellular matrix remodeling. In addition, IL-13 is required for immunity to many helminth infections. IL-13 signals via the type-II IL-4 receptor, a heterodimeric receptor of IL-13Rα1 and IL-4Rα, which is also utilized by IL-4. IL-13 also binds to IL-13Rα2, but with much higher affinity than the type-II IL-4 receptor. Binding of IL-13 to IL-13Rα2 has been shown to attenuate IL-13 signaling through the type-II IL-4 receptor. However, molecular determinants that dictate the specificity and affinity of mouse IL-13 for the different receptors are largely unknown. Here, we used high-density overlapping peptide arrays, structural modeling and molecular docking methods to map IL-13 binding sequences on its receptors. Predicted binding sequences on mouse IL-13Rα1 and IL-13Rα2 were in agreement with the reported human IL-13 receptor complex structures and site-directed mutational analysis. Novel structural differences were identified between IL-13 receptors, particularly at the IL-13 binding interface. Notably, additional binding sites were observed for IL-13 on IL-13Rα2. In addition, the identification of peptide sequences that are unique to IL-13Rα1 allowed us to generate a monoclonal antibody that selectively binds IL-13Rα1. Thus, high-density peptide arrays combined with molecular docking studies provide a novel, rapid, and reliable method to map cytokine- receptor interactions that may be used to generate signaling and decoy receptor-specific antagonists.

Simple spectrophotometric method for estimation of disodium edetate in topical gel formulations

Abstract: A simple, sensitive, cost-effective and reproducible UV-spectrophotometric method has been developed and validated for the estimation of disodium edetate in topical gel formulations. Solution of disodium edetate reacts with ferric chloride to form complex in 0.1 N HCl giving λmax at 270 nm. Beer's law was obeyed in the concentration range of 5-50 μg/mL (r (2)= 0.9997). The limit of detection and limit of quantitation were found to be 1.190 and 3.608 μg/mL, respectively. The results show that the procedure is accurate, precise, and reproducible (relative standard deviation < 1%), while being simple and less time consuming. The study concluded that the UV-spectrophotometric method could be used for the quantification of disodium edetate in pure form as well as in pharmaceutical formulations.

A simple and sensitive spectrophotometric method for estimation of diethylene triamine penta acetic acid (DTPA) in topical gel formulations

Abstract: A simple, sensitive, economical and reproducible UV-Spectrophotometric method was developed for the estimation of DTPA in topical gel formulations. The method is based on NaFeDTPA complex formation by the reaction of DTPA with ferric chloride in 0.1N HCl. Optimum conditions for the reaction were investigated and absorbance was read at λmax at 272 nm. Bears law was obeyed in the range of 5μg/mL-50μg/mL with correlation of 0.9998. The detection and quantitation limits were found to be 0.8701 and 2.6366 μg/mL respectively. The proposed method was successfully applied for the determination of DTPA in topical gel formulations. Accuracy was examined through recovery studies. The results show that the procedure is accurate, precise and reproducible (relative standard deviation < 1 %), while being simple, cost effective and less time consuming, which proves the suitability of the proposed method for the estimation of DTPA in topical gel formulations.

Toward answering time-related questions from adverse event reports using ontology-based approaches

Abstract: Identifying the time patterns from medical device adverse event reports can help clinical researchers or medical devices manufacturers to understand cause of events and predict future events. Ontologybased approaches can provide a reliable and effective system for representing both events and temporal relations, annotating the useful information from narratives, and querying and reasoning over the data. This paper discuss our vision on such a system, as well as challenges we encountered during our preliminary studies.