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A Gopala Krishna
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A Gopala Krishna
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A Gopala Krishna
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Aradhyam, Gopala K.
Aradhyam, G. K.
Krishna Aradhyam, Gopala
Aradhyam, Gopala Krishna
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4 results
Now showing 1 - 4 of 4
- PublicationChaperoning against amyloid aggregation: Monitoring in vitro and in vivo(01-01-2019)
;Vignesh, RavichandranProtein aggregation and inclusion body formation have been a key causal phenomenon behind a majority of neurodegenerative disorders. Various approaches aimed at preventing the formation/elimination of protein aggregates are being developed to control these diseases. Molecular chaperones are a class of protein that not only direct the functionally relevant fold of the protein but also perform quality control against stress, misfolding/aggregation. Genes that encode molecular chaperones are induced and expressed in response to extreme stress conditions to “salvage” the cell by the “unfolded protein response” (UPR) signaling pathway. Here we describe in detail the various in vitro and in vivo assays involved in identifying the chaperone activity of proteins using human calnuc as a model protein. Calnuc is a Golgi resident, calcium-binding protein, identified as chaperone protein and is reported to protect the cells against the cytotoxicity caused by amyloidosis and ER stress. Calnuc is also reported to regulate G αi activity and inflammation apart from the role of chaperoning against amyloid proteins. - PublicationMeasurement of intracellular Ca2 + mobilization to study GPCR signal transduction(01-01-2017)
;Ashokan, AnishaUnderstanding G protein-coupled receptor (GPCR) structure–function relationship and its activation mechanism has been broadly explored using mutational strategy due to problems in GPCR crystallization. Probing into GPCR: effector (G protein/β-arrestin) interactions and downstream signaling are important aspects of GPCR research. Among the G proteins, though there are some approaches to investigate Gq-mediated signaling, they involve the use of radioactivity and are qualitative in nature. Our method described here makes use of the cell permeable nature of fluorescent Ca2 + indicator dye, fura2AM, that binds with the Ca2 + released in response to GPCR: Gq interaction on ligand treatment. Using this spectrophotometric method, EC50 values of the GPCR: ligand binding can be calculated and the binding affinity can be analyzed. - PublicationEngineered glucose to generate a spectroscopic probe for studying carbohydrate biology(17-07-2012)
;Tripathi, Ashish ;Singh, Vibha ;Aishwarya, K. G.; Hotha, SrinivasMetabolic carbohydrate engineering by exogenously added monosaccharide supplement is a technique of importance for studying physiological role of various glycans. Additionally, it also has the potential of developing new drug molecules for specific targeting. Lack of a spectroscopic reporting moiety in carbohydrates makes understanding their biochemical and physiological role very difficult. Towards this goal, we have modified glucose, with a propargyl group, wherein an azido coumarinyl profluorophore has been linked by "click chemistry." Here, we demonstrate the uptake and incorporation of this modified monosaccharide into bacteria, yeast and mammalian cells. We show that modification at C-2 (carbon numbered 2, according to IUPAC) position is tolerated best, and uptake is only slightly lower compared to glucose. In the presence of C-2-modified glucose, growth kinetics and cellular viability were also minimally affected in all the cell types used. Fluorescence spectroscopy of the labeled biomolecule and fluorescence imaging of the cells demonstrate that C-2-modified glucose is metabolically incorporated not only in the cell membrane but also accumulates in the nucleus. Such a fluorophore, incorporated into biomolecules, can be used as a tool to understand their structure-function relationship. Here, we show that the incorporation of such a fluorophore in a carbohydrate moiety may enable studying the physiological and biochemical processes associated with membranes. © 2012 Springer Science+Business Media, LLC. - PublicationEffective purification of recombinant peptide ligands for GPCR research(01-01-2017)
;Ashokan, AnishaPeptide purification from natural sources and chemical synthesis is cumbersome with various shortcomings such as low yield, high cost of production, error prone, and restricted by nature of amino acids. Though recombinant DNA technology had overcome all these setbacks for larger proteins, it is still a challenge to produce peptides that are salt free and without impurities. Our approach discussed in this chapter deals with easy and effective purification of peptides of varying sizes (up to 10 kDa), expressed as fusion proteins in bacterial system. This includes cleavage of fusion affinity tag by “PreScission protease” in volatile buffer followed by selective acetonitrile precipitation of high-molecular-weight tag in order to purify peptides in solution. This method can be used to purify peptides in large scale for various biochemical and physiological studies.