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    3D model of PSI-LHCI supercomplexes from chlamydomonas reinhardtii
    ( 2013-01-01) Yadavalli, Venkateswarlu ; Malleda, Chandramouli ; Subramanyam, Rajagopal
    The function of Photosystem I (PSI) is catalyzing one of the initial steps in driving oxygenic photosynthesis in cyanobacteria, algae and higher plants. The recent crystallographic model at 3.3 Å resolution represents the most complete plant PSI structure. The Chlamydomonas reinhardtii PSI-LHCI supercomplex structure is not known since it contains a unique structure having additional subunits of light harvesting complex I (LHCI). We have modeled PSI core and LHCI in order to elucidate the structure of PSI-LHCI supercomplexes of C. reinhardtii. Most of the core subunits are homologous to the higher plants except PsaO and now it has been modeled based on threading. All core subunits were located similarly like pea structure however, PsaO, a new subunit is closely located to the PsaH, PsaI and PsaL subunits. The location of PsaO subunit at this position may suggest that it may be involved in state transition mechanism in C. reinhardtii. From our model, it indicates that there are non-covalent strong inter protein-protein relationship, especially from PSI core to LHCI. Our 3D model may give the structural information for better understanding of PSI-LHCI arrangement and its physiological role in C. reinhardtii as well as other algae, where it serves many biotechnological applications.
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    4-Nonylphenol-enhanced EZH2 and RNF2 expression, H3K27me3 and H2AK119ub1 marks resulting in silencing of p21 < sup > CDKN1A < /sup > in vitro
    ( 2019-06-01) Ghosh, Krishna ; Chatterjee, Biji ; Maheswari, Uma ; Athifa, Mariyam ; Kanade, Santosh R.
    Aim: To examine the impact of 4-nonylphenol (4-NP), on the expression of polycomb repressive complexes and cellular proliferation. Materials & methods: Cell proliferation assays, quantitative PCR, Western blotting, luciferase reporter assay, chromatin immunoprecipitation-quantitative PCR were used for the study. Results: The 4-NP at 100 nM concentration significantly increased proliferation of MCF-7 cells. It enhanced the expression of RNF2-BMI1 and EZH2-SUZ12 and concomitantly increased H2AK119ub1 and H3K27me3 repressive marks at p21 proximal promoter resulting in its reduced expression. Selective inhibition of RNF2 or EZH2 reverted the 4-NP action. The phospho-CREB, SP1 and E2F-1 are enriched at proximal promoter of RNF2 and EZH2 and cyclin D1, but not p21. Conclusion: The 4-NP-mediated upregulation of RNF2 and EZH2 resulted in epigenetic silencing of p21.
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    5‐Aminolevulinic Acid: A Potential Herbicide/Insecticide from Microorganisms
    ( 1994-01-01) Sasikala, Ch ; Ramana, Ch V. ; Rao, P. Raghuveer
    5‐Aminolevulinic acid (ALA), an intermediate of the biological tetrapyrrole synthesis, can be used as a photodynamic herbicide/insecticide. Among the various microorganisms capable of its production, anoxygenic phototrophic bacteria produce ALA in considerable amounts, making it worthwhile to work toward commercial exploitation. Knowledge of the biochemical synthesis of ALA and its physiological and genetic regulation in microorganisms can enable the biotechnologist to manipulate them for enhancing ALA production for possible practical applications. Copyright © 1994 American Institute of Chemical Engineers (AIChE)
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    A carbohydrate binding module-5 is essential for oxidative cleavage of chitin by a multi-modular lytic polysaccharide monooxygenase from Bacillus thuringiensis serovar kurstaki
    ( 2019-04-15) Manjeet, Kaur ; Madhuprakash, Jogi ; Mormann, Michael ; Moerschbacher, Bruno M. ; Podile, Appa Rao
    Conversion of crystalline chitin to soluble sugar molecules, using lytic polysaccharide mono-oxygenases (LPMOs) has emerged as a new avenue for the production of biofuels. The present study describes the role of accessory domains in a multi-modular LPMO from Bacillus thuringiensis serovar kurstaki (BtLPMO10A). The full length BtLPMO10A (BtLPMO10A-FL) possesses an N-terminal LPMO of AA10 family (BtAA10) and a C-terminal CBM5 (BtCBM5) connected via two fibronectin (Fn) III domains (aligned as AA10-FnIII-FnIII-CBM5 from N- to C-terminus). To determine the role of individual domains, we generated truncation mutants of BtLPMO10A-FL. Substrate binding and kinetic studies revealed that BtCBM5 was involved in increasing binding efficiency of BtAA10 which otherwise has feeble binding towards β-chitin and could not bind to α-chitin. Furthermore, binding assays also indicated that the presence of CBM5 increases the binding efficiency of BtLPMO10A-FL under extreme pH conditions. FnIII domains neither bind nor assist BtLPMO10A-FL in chitin binding and serve as linkers in BtLPMO10A-FL. BtLPMO10A-FL and BtAA10 generated oxidized chito-oligosaccharides from the insoluble β-chitin substrate. It is concluded that BtCBM5 is responsible for increasing binding efficiency of BtLPMO10A-FL, whereas; BtAA10 domain is accountable for oxidative cleavage of recalcitrant chitin.
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    A comparative binding mechanism between human serum albumin and α-1-acid glycoprotein with corilagin: Biophysical and computational approach
    ( 2016-01-01) Yeggoni, Daniel Pushparaju ; Rachamallu, Aparna ; Subramanyam, Rajagopal
    The binding of corilagin with plasma serum proteins like human serum albumin (HSA) and α-1-acid glycoprotein (AGP) was investigated under physiological conditions. To understand the pharmacological importance of the corilagin molecule, anti-inflammatory activity on mouse macrophages (RAW 264.7) cell lines was studied. This study reveals that corilagin caused an increase in inhibition growth of inflamed macrophages in concentration-dependent manner with an IC50 value of 66 μM. Further, intrinsic fluorescence of HSA and AGP was quenched upon titration of corilagin, and the binding constants obtained from fluorescence emission was found to be Kcorilagin 4.2 ± 0.02 × 105 M-1 which corresponds to the free energy of -7.6 kcal M-1 at 25°C for a HSA-corilagin complex. Interestingly, corilagin showed binding with AGP, an acute phase protein, and the binding constant was found to be Kcorilagin = 1.5 ± 0.01 × 104 M-1 and its free energy was -5.6 kcal M-1 at 25°C. Further, the average binding distance, r, between the donor (HSA) and acceptor (corilagin) was calculated and found to be 1.32 nm according to Förster's theory of non-radiation energy transfer. Later, circular dichroism studies emphasized that there are marginal changes in secondary structural conformation of HSA in the presence of corilagin. Corilagin is specifically bound to site I of HSA which was proved by site specific marker, phenylbutazone. Furthermore, the binding details between corilagin and HSA revealed that corilagin was bound to subdomain IIA through multiple interactions like hydrogen bonding and hydrophobic effects. Molecular dynamic studies (MD) also suggest that binding is very precise to site I (IIA domain) on HSA. Also, MD studies showed that HSA-corilagin complex reaches equilibration state at around 4 ns, which proves that the HSA-corilagin complex is stable in nature, hence the experimental and computational results are in agreement. Thus, examining the interaction mechanism of corilagin with plasma proteins may play a critical role in developing corilagin inspired drugs.
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    A comprehensive study on core enzymes involved in starch metabolism in the model nutricereal, foxtail millet (Setaria italica L.)
    ( 2021-01-01) Dhaka, Annvi ; Muthamilarasan, Mehanathan ; Prasad, Manoj
    Starch biosynthesis is an important process in plants as starch serves as a source of carbon and energy. In cereals, starch is the predominant constituent of the grains that provide carbohydrates in food and feed. Given its importance, the biosynthesis and accumulation of starch have been well studied in major cereals. However, in millets, no such study provides insights into the starch biosynthesis and diversity of enzymes involved in this process. In foxtail millet (Setaria italica), we have identified and characterized six classes of enzyme-encoding genes involved in starch metabolism, viz., ADP glucose phosphorylase, starch synthase, starch branching enzyme, starch debranching enzyme, phosphorylase, and disproportionating enzyme. Analysis of gene structure, chromosomal localization, phylogenetic analysis, and study of domain composition were performed to gain insights into the structure and organization of these gene families. Further, expression profiling of these genes in two cultivars contrastingly differing in grain amylose content was performed at different seed development stages. The expression data showed spatiotemporally divergent expression patterns of the genes and pinpointed several candidate genes that could be targeted for further functional characterization to study the starch metabolism in millets as well as to improve starch content through genomics approaches.
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    A dominant mutation in the light-oxygen and voltage2 domain vicinity impairs phototropin1 signaling in tomato
    ( 2014-01-01) Sharma, Sulabha ; Kharshiing, Eros ; Srinivas, Ankanagari ; Zikihara, Kazunori ; Tokutomi, Satoru ; Nagatani, Akira ; Fukayama, Hiroshi ; Bodanapu, Reddaiah ; Behera, Rajendra K. ; Sreelakshmi, Yellamaraju ; Sharma, Rameshwar
    In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A′α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A′α helix region in phototropic signaling of tomato. © 2014 American Society of Plant Biologists. All rights reserved.
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    A dominant mutation in the light-oxygen and voltage2 domain vicinity impairs phototropin1 signaling in tomato
    ( 2014-01-01) Sharma, Sulabha ; Kharshiing, Eros ; Srinivas, Ankanagari ; Zikihara, Kazunori ; Tokutomi, Satoru ; Nagatani, Akira ; Fukayama, Hiroshi ; Bodanapu, Reddaiah ; Behera, Rajendra K. ; Sreelakshmi, Yellamaraju ; Sharma, Rameshwar
    In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A′α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A′α helix region in phototropic signaling of tomato. © 2014 American Society of Plant Biologists. All rights reserved.
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    A fucose specific lectin from Aspergillus flavus induced interleukin-8 expression is mediated by mitogen activated protein kinase p38
    ( 2017-04-01) Ghufran, Md Sajid ; Ghosh, Krishna ; Kanade, Santosh R.
    Aspergillus flavus is an ubiquitous, opportunistic fungus responsible to cause invasive fungal allergic diseases, including bronchopulmonary invasive aspergillosis in persons with altered immune function. Lectins have been implicated as interaction mediators between the pathogenic fungi and human host. We isolated L-fucose specific lectin from A. flavus (FFL) and purified it to homogeneity with a combination of ion exchange and hydrophobic interaction chromatography methods. Its hemagglutination activity was significantly inhibited by 125 μM L-fucose as compared to other sugars and sugar derivatives. We, then used human cell line L-132, and U937 cell line to explore the possible cytotoxicity and proinflammatory effect of this fucose-specific lectin. The lectin induced the expression of proinflammatory cytokine interleukin-8 (IL-8) in a dose-dependent manner, and it was found to be associated with the p38 mitogen activated protein kinase (MAPK). The p38MAPK signalling pathway regulates the transcription factor activator protein-1 (AP-1) activity, which is the integration point of many signals that can differentially affect the expression and transcriptional activity of a cell. We observed activation of c-Jun, a critical component of the AP-1 complex, mediated by p38MAPK upon the FFL treatment in L-132 cells. Finally, inhibition of p38MAPK by a specific inhibitor attenuates the c-Jun, suggesting the p38MAPK involvement in the c-Jun activation, which in turn transcriptionally activates the induction of IL-8 in response to the lectin. Thus, this study showed a potential lectin-mediated mechanism to modulate the immune response during host-fungus interactions.
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    A low-cost high-throughput method for plant genomic DNA isolation
    ( 2020-01-01) Gupta, Prateek ; Salava, Hymavathi ; Sreelakshmi, Yellamaraju ; Sharma, Rameshwar
    Many of the functional genomics methods require isolation of genomic DNA from large population of plants. The selection of DNA isolation protocols depends on several factors such as choice of starting material, ease of handling, time and labor required for isolation, the final quantity as well as the quality of genomic DNA. We outline here a high-throughput method of DNA extraction from different plant species including cereal crops. The protocol can be used for extraction of DNA in single tubes as well as for large formats in 96-well plates. The protocol includes steps for eliminating interfering secondary products such as phenolics. This protocol can be applied for high-throughput isolation of DNA for varied applications such as TILLING, mapping, fingerprinting, etc. as a cost-effective protocol compared to commercial kits.
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    A low-cost high-throughput method for plant genomic DNA isolation
    ( 2020-01-01) Gupta, Prateek ; Salava, Hymavathi ; Sreelakshmi, Yellamaraju ; Sharma, Rameshwar
    Many of the functional genomics methods require isolation of genomic DNA from large population of plants. The selection of DNA isolation protocols depends on several factors such as choice of starting material, ease of handling, time and labor required for isolation, the final quantity as well as the quality of genomic DNA. We outline here a high-throughput method of DNA extraction from different plant species including cereal crops. The protocol can be used for extraction of DNA in single tubes as well as for large formats in 96-well plates. The protocol includes steps for eliminating interfering secondary products such as phenolics. This protocol can be applied for high-throughput isolation of DNA for varied applications such as TILLING, mapping, fingerprinting, etc. as a cost-effective protocol compared to commercial kits.
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    A new chitinase-D from a plant growth promoting Serratia marcescens GPS5 for enzymatic conversion of chitin
    ( 2016-11-01) Vaikuntapu, Papa Rao ; Rambabu, Samudrala ; Madhuprakash, Jogi ; Podile, Appa Rao
    The current study describes heterologous expression and biochemical characterization of single-modular chitinase-D from Serratia marcescens (SmChiD) with unprecedented catalytic properties which include chitobiase and transglycosylation (TG) activities besides hydrolytic activity. Without accessory domains, SmChiD, hydrolyzed insoluble polymeric chitin substrates like colloidal, α- and β-chitin. Activity studies on CHOS with degree of polymerization (DP) 2–6 as substrate revealed that SmChiD hydrolyzed DP2 with a chitobiase activity and showed TG activity on CHOS with DP3–6, producing longer chain CHOS. But, the TG products were further hydrolyzed to shorter chain CHOS with DP1–2 products. SmChiD with its unique catalytic properties, could be a potential enzyme for the production of long chain CHOS and also for the preparation of efficient enzyme cocktails for chitin degradation.
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    A novel aldo-keto reductase from Jatropha curcas L. (JcAKR) plays a crucial role in the detoxification of methylglyoxal, a potent electrophile
    ( 2016-05-20) Mudalkar, Shalini ; Sreeharsha, Rachapudi Venkata ; Reddy, Attipalli Ramachandra
    Abiotic stress leads to the generation of reactive oxygen species (ROS) which further results in the production of reactive carbonyls (RCs) including methylglyoxal (MG). MG, an α, β-dicarbonyl aldehyde, is highly toxic to plants and the mechanism behind its detoxification is not well understood. Aldo-keto reductases (AKRs) play a role in detoxification of reactive aldehydes and ketones. In the present study, we cloned and characterised a putative AKR from Jatropha curcas (JcAKR). Phylogenetically, it forms a small clade with AKRs of Glycine max and Rauwolfia serpentina. JcAKR was heterologously expressed in Escherichia coli BL-21(DE3) cells and the identity of the purified protein was confirmed through MALDI-TOF analysis. The recombinant protein had high enzyme activity and catalytic efficiency in assays containing MG as the substrate. Protein modelling and docking studies revealed MG was efficiently bound to JcAKR. Under progressive drought and salinity stress, the enzyme and transcript levels of JcAKR were higher in leaves compared to roots. Further, the bacterial and yeast cells expressing JcAKR showed more tolerance towards PEG (5%), NaCl (200 mM) and MG (5 mM) treatments compared to controls. In conclusion, our results project JcAKR as a possible and potential target in crop improvement for abiotic stress tolerance.
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    A novel L-fucose-binding lectin from Fenneropenaeus indicus induced cytotoxicity in breast cancer cells Biji Chatterjee
    ( 2017-01-01) Ghosh, Krishna ; Yadav, Nitin ; Kanade, Santosh R.
    Lectins are omnipresent in almost all life forms, being the proteins which specifically bind to carbohydrate moieties on the cell surface; they have been explored for their anti-tumour activities. In this study, we purified a fucose specific-lectin (IFL) from Fenneropenaeus indicus haemolymph using fucose-affinity column and characterized for its haemagglutination activity, carbohydrate specificity, dependency on cations and cytotoxicity against cancer cells. The lectin showed nonspecificity against human erythrocytes. It was a Ca2+-dependent lectin which remained stable over wide pH and temperature ranges. The lectin showed effective dose dependent cytotoxicity against different human cancer cell lines and induced apoptosis in MCF- 7 cells as evidenced by DNA ladder assay and PARP cleavage in a dose dependent manner. Moreover, an increased p21 level corresponding to cyclin D downregulation in response to IFL treatment was observed which might work as probable factors to inhibit cell growth and induce apoptosis of MCF-7 cells. Therefore, we report a novel lectin from the prawn haemolymph with high specificity for L-fucose and antiproliferative towards human cancer cells. However, further establishment of the modus operandi of this lectin is required to enable its biotechnological applications.
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    A novel method of measuring volume changes of mesophyll cell protoplasts and the effect of mercuric chloride on their osmotically-induced swelling
    ( 1999-01-01) Willmer, C. M. ; Padmasree, K. ; Raghavendra, A. S.
    A quick and accurate method of monitoring changes of volume of mesophyll cell protoplasts (MCP) of pea was devised using the difference in absorbance of the protoplasts at 440 nm and 750 nm (OD 440-750); when protoplasts expanded in response to changing the external medium from 0.4 M sorbitol to 0.3 M sorbitol OD 440-750 values increased and, conversely, when protoplasts were transferred from 0.4 to 0.5 M sorbitol, protoplasts contracted. The kinetics of expansion or contraction of the protoplasts could also be monitored using this method and the half-time for water exchange for expanding protoplasts (about 10 s) was slightly higher than that for contracting protoplasts. A study of the effects of the water channel blocker, mercuric chloride, on swelling protoplasts showed that 500 μM irreversibly damaged protoplasts, 5-10 μM had a negligible inhibitory effect on swelling while 100 μM had a large inhibitory effect often completely inhibiting swelling. A preliminary study indicated that mercapto-ethanol reversed the inhibitory effect of mercuric chloride on protoplast swelling.
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    A phytoalexin is modified to less fungitoxic substances by crown rot pathogen in groundnut Arachis hypogaea L.
    ( 1998-06-01) Sailaja, P. R. ; Podile, A. R.
    Phytoalexins from four different treatments viz. control, AF 1-treated, A. niger-treated, and dual inoculated were separated by TLC showed that one phytoalexin with an Rf value of 0.628 (P1) appeared on 2nd day only in dual-inoculated seeds of groundnut (A. hypogaea ). By 3rd day three additional phytoalexins were visualized in response to A. niger-treatment with lower Rf values 0.485 (P2), 0.388 (P3) and 0.314 (P4) as compared to P1. In dual inoculated seedlings, P1 and P3 could be visualized while only P1 appeared in response to AF 1 on 3rd day. All the compounds lost fluorescence on exposure to light, got converted to pale yellow colour. In all the treatments no phytoalexin accumulation was observed after 3rd day. All the four phytoalexins had a major peak between 338 and 339.5 nm. In potato dextrose broth, the growth of A. niger showed a steady increase up to 32 hr while it was significantly inhibited with P1 in microtiter plates. P2, P3 and P4 (in the same order) had significantly less antifungal activity as compared to P1. The antifungal activity of the phytoalexins decreased with decrease in their Rf value.
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    A proteasome inhibitor-stimulated Nrf1 protein-dependent compensatory increase in proteasome subunit gene expression reduces polycomb group protein level
    ( 2012-10-19) Balasubramanian, Sivaprakasam ; Kanade, Santosh ; Han, Bingshe ; Eckert, Richard L.
    The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study, we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132.Weshow that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G2, and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a prosurvival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells and confirm that PcG protein levels are regulated by proteasome activity. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
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    A rapid and sensitive method for determination of carotenoids in plant tissues by high performance liquid chromatography
    ( 2015-02-06) Gupta, Prateek ; Sreelakshmi, Yellamaraju ; Sharma, Rameshwar
    Background: The dietary carotenoids serve as precursor for vitamin A and prevent several chronic-degenerative diseases. The carotenoid profiling is necessary to understand their importance on human health. However, the available high-performance liquid chromatography (HPLC) methods to resolve the major carotenoids require longer analysis times and do not adequately resolve the violaxanthin and neoxanthin. Results: A fast and sensitive HPLC method was developed using a C30 column at 20°C with a gradient consisting of methanol, methyl-tert-butyl ether and water. A total of 15 major carotenoids, including 14 all-trans forms and one cis form were resolved within 20 min. The method also distinctly resolved violaxanthin and neoxanthin present in green tissues. Additionally this method also resolved geometrical isomers of the carotenoids. Conclusion: The HPLC coupled with C30 column efficiently resolved fifteen carotenoids and their isomers in shorter runtime of 20 min. Application of this method to diverse matrices such as tomato fruits and leaves, Arabidopsis leaves and green pepper fruits showed the versatility and robustness of the method. The method would be useful for high throughput analysis of large number of samples.
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    A rapid and sensitive method for determination of carotenoids in plant tissues by high performance liquid chromatography
    ( 2015-02-06) Gupta, Prateek ; Sreelakshmi, Yellamaraju ; Sharma, Rameshwar
    Background: The dietary carotenoids serve as precursor for vitamin A and prevent several chronic-degenerative diseases. The carotenoid profiling is necessary to understand their importance on human health. However, the available high-performance liquid chromatography (HPLC) methods to resolve the major carotenoids require longer analysis times and do not adequately resolve the violaxanthin and neoxanthin. Results: A fast and sensitive HPLC method was developed using a C30 column at 20°C with a gradient consisting of methanol, methyl-tert-butyl ether and water. A total of 15 major carotenoids, including 14 all-trans forms and one cis form were resolved within 20 min. The method also distinctly resolved violaxanthin and neoxanthin present in green tissues. Additionally this method also resolved geometrical isomers of the carotenoids. Conclusion: The HPLC coupled with C30 column efficiently resolved fifteen carotenoids and their isomers in shorter runtime of 20 min. Application of this method to diverse matrices such as tomato fruits and leaves, Arabidopsis leaves and green pepper fruits showed the versatility and robustness of the method. The method would be useful for high throughput analysis of large number of samples.
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    A rapid, efficient, and low-cost BiFC protocol and its application in studying in vivo interaction of seed-specific transcription factors, RISBZ and RPBF
    ( 2021-11-01) Thakur, Tanika ; Gandass, Nishu ; Mittal, Kajal ; Jamwal, Pallavi ; Muthamilarasan, Mehanathan ; Salvi, Prafull
    Proteins regulate cellular and biological processes in all living organisms. More than 80% of the proteins interact with one another to perform their respective functions; therefore, studying the protein–protein-interaction has gained attention in functional characterization studies. Bimolecular fluorescence complement (BiFC) assay is widely adopted to determine the physical interaction of two proteins in vivo. Here, we developed a simple, yet effective BiFC assay for protein–protein-interaction using transient Agrobacterium-mediated-transformation of onion epidermal cells by taking case study of Rice-P-box-Binding-Factor (RPBF) and rice-seed-specific-bZIP (RISBZ) in vivo interaction. Our result revealed that both the proteins, i.e., RISBZ and RPBF, interacted in the nucleus and cytosol. These two transcription factors are known for their coordinate/synergistic regulation of seed-protein content via concurrent binding to the promoter region of the seed storage protein (SSP) encoding genes. We further validated our results with BiFC assay in Nicotiana by agroinfiltration method, which exhibited similar results as Agrobacterium-mediated-transformation of onion epidermal cells. We also examined the subcellular localization of RISBZ and RPBF to assess the efficacy of the protocol. The subcellular localization and BiFC assay presented here is quite easy-to-follow, reliable, and reproducible, which can be completed within 2–3 days without using costly instruments and technologies that demand a high skill set.