The Experts below are selected from a list of 1539 Experts worldwide ranked by ideXlab platform
Anjali A Karande - One of the best experts on this subject based on the ideXlab platform.
-
correlation of Abrin mediated inhibition of protein synthesis and apoptosis
Iubmb Life, 2019Co-Authors: Vinita Tiwari, Anjali A KarandeAbstract:The plant toxin, Abrin, a type-II ribosome inactivating protein, is extremely lethal, the human fatal dose being similar to 1 mu g/kg body weight. Abrin has been classified as an agent for bioterrorism, which is of concern. Conversely, the high toxic property of Abrin has been employed in generating immunotoxins, whereas its toxin moiety is conjugated to cell surface marker-specific antibodies for cell-targeted killing. Different cell types exhibit variable levels of sensitivity to Abrin toxicity; therefore, adequate knowledge of the molecular mechanism that governs the activity of the protein would be a safeguard. To gain insights into this, two cell lines requiring strikingly different concentrations of Abrin for inactivating ribosomes were studied. Employing conjugates of the wild-type and active site mutant of Abrin A chain with the ricin B chain, it was found that Abrin-induced apoptosis was dependent on inhibition of protein synthesis (PSI) leading to ER-stress in Ovcar-3 cells, but not in KB cells. Abrin was also observed to cause direct DNA damage in KB cells, while in Ovcar-3 cells Abrin-induced DNA damage was found to be dependent on caspases. Overall, the study demonstrates that the correlation of Abrin-mediated PSI and apoptosis is cell-specific and Abrin can induce more than one pathway to cause cell death. (c) 2018 IUBMB Life, 71(3):357-363, 2019
-
Correlation of Abrin‐mediated inhibition of protein synthesis and apoptosis
IUBMB life, 2018Co-Authors: Vinita Tiwari, Anjali A KarandeAbstract:The plant toxin, Abrin, a type-II ribosome inactivating protein, is extremely lethal, the human fatal dose being similar to 1 mu g/kg body weight. Abrin has been classified as an agent for bioterrorism, which is of concern. Conversely, the high toxic property of Abrin has been employed in generating immunotoxins, whereas its toxin moiety is conjugated to cell surface marker-specific antibodies for cell-targeted killing. Different cell types exhibit variable levels of sensitivity to Abrin toxicity; therefore, adequate knowledge of the molecular mechanism that governs the activity of the protein would be a safeguard. To gain insights into this, two cell lines requiring strikingly different concentrations of Abrin for inactivating ribosomes were studied. Employing conjugates of the wild-type and active site mutant of Abrin A chain with the ricin B chain, it was found that Abrin-induced apoptosis was dependent on inhibition of protein synthesis (PSI) leading to ER-stress in Ovcar-3 cells, but not in KB cells. Abrin was also observed to cause direct DNA damage in KB cells, while in Ovcar-3 cells Abrin-induced DNA damage was found to be dependent on caspases. Overall, the study demonstrates that the correlation of Abrin-mediated PSI and apoptosis is cell-specific and Abrin can induce more than one pathway to cause cell death. (c) 2018 IUBMB Life, 71(3):357-363, 2019
-
a chimeric protein of Abrin and abrus precatorius agglutinin that neutralizes Abrin mediated lethality in mice
Toxicon, 2017Co-Authors: Vinita Tiwari, Shradha Bagaria, Anjali A KarandeAbstract:Abstract Abrin, a type II ribosome inactivating protein from the Abrus precatorius plant, is extremely toxic. It has been shown to be 75 times more potent than its infamous sister toxin, ricin and their potential use in bio-warfare is a cause of major concern. Although several vaccine candidates are under clinical trials for ricin, none are available against Abrin. The present study proposes a chimeric protein, comprising of 1–123 amino acids taken from the A chain of Abrin and 124–175 amino acids from Abrus precatorius agglutinin A chain, as a vaccine candidate against Abrin intoxication. The design was based on the inclusion of the immunogenic region of the full length protein and the minimal essential folding domains required for inducing neutralizing antibody response. The chimera also contains the epitope for the only two neutralizing antibodies; D6F10 and A7C4, reported against Abrin till now. Active immunization with the chimera protected all the mice challenged with 45 X LD 50 of Abrin. Also, passive transfer of antibodies raised against the chimera rescued all mice challenged with 50 X LD 50 of toxin. Hence the chimeric protein appears to be a promising vaccine candidate against Abrin induced lethality.
-
A monoclonal antibody to an Abrin chimera recognizing a unique epitope on Abrin A chain confers protection from Abrin-induced lethality.
Human vaccines & immunotherapeutics, 2015Co-Authors: Meenakshi Sundaram Kumar, Anjali A KarandeAbstract:Abrin, obtained from the seeds of Abrus precatorius plant, is a potent toxin belonging to the family of type II ribosome-inactivating proteins. Recently, a recombinant vaccine consisting of the A subunits of Abrin and its homolog Abrus precatorius agglutinin (APA) was demonstrated to protect mice from Abrin lethality. Toward identifying neutralizing epitopes recognized during this response, we generated monoclonal antibodies against the proposed vaccine candidate. One antibody, namely A7C4, the corresponding epitope of which was found to be distal to the active site of the enzymatic A chain, prevented Abrin-mediated toxicity on cells and Abrin-induced lethality in mice but did not inhibit the catalytic activity of the A chain. The in vivo protection conferred by monoclonal antibody A7C4 highlights the potential use of this antibody as a promising immunotherapeutic.
-
Inhibition of protein synthesis leading to unfolded protein response is the major event in Abrin-mediated apoptosis
Molecular and Cellular Biochemistry, 2015Co-Authors: Ritu Mishra, Meenakshi Sundaram Kumar, Anjali A KarandeAbstract:Abrin obtained from the plant Abrus precatorius inhibits protein synthesis and also triggers apoptosis in cells. Previous studies from our laboratory suggested a link between these two events. Using an active site mutant of Abrin A-chain which exhibits 225-fold lower protein synthesis inhibitory activity than the wild-type Abrin A-chain, we demonstrate in this study that inhibition of protein synthesis induced by Abrin is the major factor triggering unfolded protein response leading to apoptosis. Since Abrin A-chain requires the B-chain for internalization into cells, the wild-type and mutant recombinant Abrin A-chains were conjugated to native ricin B-chain to generate hybrid toxins, and the toxic effects of the two conjugates were compared. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild-type ricin B-rABRA conjugate as expected. The mutant conjugate activated p38MAPK and caspase-3 similar to its wild-type counterpart although at later time points. Overall, these results confirm that inhibition of protein synthesis is the major event contributing to Abrin-mediated apoptosis.
S. K. Podder - One of the best experts on this subject based on the ideXlab platform.
-
Calorimetric studies on the stability of the ribosome-inactivating protein Abrin II: effects of pH and ligand binding
Biochemical Journal, 1999Co-Authors: Jayarapu Krupakar, Chittoor P. Swaminathan, Puspendu K. Das, Avadhesha Surolia, S. K. PodderAbstract:The effects of pH and ligand binding on the stability of Abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (Tm) 319.2K] and the A-subunit (Tm 324.6K) of Abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of Abrin II is similar to that of the higher-temperature transition. However, its Tm is 2.4K lower than that of the higher-temperature peak of intact Abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the Tm values as well as the enthalpies of both transitions. This effect is more pronounced at pH7.2 than at pH4.5. This suggests that ligand binding stabilizes the native conformation of Abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of Abrin II at pH7.2. The presence of two binding sites for lactose on the Abrin II molecule is also indicated by isothermal titration calorimetry. Plotting DeltaHm (the molar transition enthalpy at Tm) against Tm yielded values for DeltaCp (change in excess heat capacity) of 27+/-2 kJ.mol-1.K-1 for the B-subunit and 20+/-1 kJ.mol-1.K-1 for the A-subunit. These values have been used to calculate the thermal stability of Abrin II and to surmise the mechanism of its transmembrane translocation.
-
A- and B-subunit variant distribution in the holoprotein variants of protein toxin Abrin: variants of Abrins I and III have constant toxic A subunits and variant lectin B subunits.
Archives of Biochemistry and Biophysics, 1997Co-Authors: Ramesh Hegde, S. K. PodderAbstract:Abstract The cytotoxic lectin Abrin shows more than 30 variant forms (R. Hegde, T. K. Maiti, and S. K. Podder, 1991, Anal. Biochem. 194, 101–109). The lectin B subunit as cause for variance in Abrins I and III was detected by a combination of one- and two-dimensional electrophoresis and Western blotting. Intriguingly, in Abrin I but not in Abrin III, association of a single A subunit with the variant B subunits shifts the holoprotein p I toward the alkaline side indicating that the subunit association involves neutralization of few negative charges. The B-subunit variants of Abrins I and III overlap in their p I , and the A-subunit association gives the holoproteins a distinctness on isoelectric focusing gel. The results were also confirmed by analyzing the pH titration curves. These differences in the subunit association pattern between Abrins I and III are in corroboration with the previously observed differences in the kinetics of protein synthesis inactivation and accessibility of the disulfide bridge to reducing agents in the presence or absence of putative receptor (R. Hegde, A. Karande, and S. K. Podder, 1993 Eur. J. Biochem. 215, 411–419). Further, the genetic origin of variance was confirmed by peptide mapping of the individual subunit variants. Considering a theoretical value of 0.1 to 0.2 p I /charge, a 15–17 charge difference could be predicted between the variants of two extreme p I s. The fact that the A subunits are not shared between the groups was taken to interpret that the protein synthesized as prepro form is processed posttranslationally and the processing takes place only after the disulfide bond formation between A and B subunits. The N-terminal 16 amino acids of A subunits of Abrins I and III showed 26% dissimilarity. The A subunits of Abrins I and III did not react with concanavalin A, indicating that the heterogeneity in the molecular weight is because of differential processing but not because of glycosylation.
-
The variants of the protein toxins Abrin and ricin. A useful guide to understanding the processing events in the toxin transport.
European journal of biochemistry, 1993Co-Authors: Ramesh Hegde, Anjali A Karande, S. K. PodderAbstract:Kinetic data on inhibition of protein synthesis in thymocyte by three Abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by k1-versus-concentration plots were Abrin II, III > ricin > Abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of Abrin I, II, III and ricin, respectively. To account for the observed differences in the dose-dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low-pH-induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that Abrin-I (4.13 × 105 M−1 s−1) association with putative receptor (4-methylumbelliferyl-α-D-galactoside) is nearly two times more often than Abrin III (2.6 × 105 M−1 s−1) at 20°C. Equillibrium binding constants of Abrin I and II to thymocyte at 37°C were 2.26 × 107 M−1 and 2.8 × 107 M−1 respectively. pH-induced structural alterations as studied by a parallel enhancement in 8-anilino-L-naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration-independent lag time (minimum lag of 41–42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose-dependent lag time is causally related to the proposed processing event. The rates of reduction of inter-subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of Abrin I is fourfold faster than that of Abrin II at pH 7.2. The rate of disulfide reduction in Abrin I could be decreased 1 I-fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose-dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.
-
purification and characterization of three toxins and two agglutinins from abrus precatorius seed by using lactamyl sepharose affinity chromatography
Analytical Biochemistry, 1991Co-Authors: Ramesh Hegde, Tapas K Maiti, S. K. PodderAbstract:Abstract Three toxins, Abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. Abrin-I did not bind on DEAE-Sephacel column chromatography and the bound Abrin-II, Abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are Abrin-I, 64,000; Abrin-II and Abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms−1 at a 0.63 n m concentration of each of Abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic Abrin-I and the highly toxic Abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.
Jiancheng Yang - One of the best experts on this subject based on the ideXlab platform.
-
Trace level detections of Abrin with high SNR piezoresistive cantilever biosensor
Sensors and Actuators B: Chemical, 2015Co-Authors: Rui Zhao, Yongzheng Wen, Jiancheng YangAbstract:Abstract In this paper, sensitive detections on Abrin are demonstrated in real-time by piezoresistive (PZR) cantilever sensors. With attempts to optimize the design and fabrication of the PZR cantilevers, the voltage fluctuation of the PZR sensors was decreased to 1 μV and the signal to noise ratio (SNR) was increased significantly. By using the biotin–streptavidin functionalized cantilevers, sensitive detections on Abrin were realized. A linear relation between the concentration of Abrin and the output voltage of PZR sensors was established within the range of 0.08–40 ng/mL (1.2–600 pM) and a limit of detection (LOD) of 80 pg/mL (1.2 pM) was obtained in PBS buffer. Meanwhile, detections on orange juice and skim milk samples spiked with Abrin were also performed, and the detection sensitivity decreased by 6–17% compared with the one in PBS buffer at the same concentrations. At last, a model of pseudo-first-order kinetics was used to analyze the interaction mechanism of Abrin and biotinylated polyclone antibody (bio-PcAb) against Abrin, which was consistent with the detection results. The proposed PZR cantilever sensors provide an economical platform for on-site detections of food contaminants with the advantages of high SNR, portability, and rapid response.
Yu Xiaomei - One of the best experts on this subject based on the ideXlab platform.
-
Trace level detections of Abrin with high SNR piezoresistive cantilever biosensor
sensors and actuators b chemical, 2015Co-Authors: Zhao Ruo, Ma Wei, Wen Yongzheng, Yang Jiancheng, Yu XiaomeiAbstract:In this paper, sensitive detections on Abrin are demonstrated in real-time by piezoresistive (PZR) cantilever sensors. With attempts to optimize the design and fabrication of the PZR cantilevers, the voltage fluctuation of the PZR sensors was decreased to 1 mu V and the signal to noise ratio (SNR) was increased significantly. By using the biotin-streptavidin functionalized cantilevers, sensitive detections on Abrin were realized. A linear relation between the concentration of Abrin and the output voltage of PZR sensors was established within the range of 0.08-40 ng/mL (1.2-600 pM) and a limit of detection (LOD) of 80 pg/mL (1.2 pM) was obtained in PBS buffer. Meanwhile, detections on orange juice and skim milk samples spiked with Abrin were also performed, and the detection sensitivity decreased by 6-17% compared with the one in PBS buffer at the same concentrations. At last, a model of pseudo-first-order kinetics was used to analyze the interaction mechanism of Abrin and biotinylated polyclone antibody (bio-PcAb) against Abrin, which was consistent with the detection results. The proposed PZR cantilever sensors provide an economical platform for on-site detections of food contaminants with the advantages of high SNR, portability, and rapid response. (C) 2015 Elsevier B.V. All rights reserved.Chemistry, AnalyticalElectrochemistryInstruments & InstrumentationSCI(E)EI0ARTICLEyuxm@pku.edu.cn112-11921
Ramesh Hegde - One of the best experts on this subject based on the ideXlab platform.
-
A- and B-subunit variant distribution in the holoprotein variants of protein toxin Abrin: variants of Abrins I and III have constant toxic A subunits and variant lectin B subunits.
Archives of Biochemistry and Biophysics, 1997Co-Authors: Ramesh Hegde, S. K. PodderAbstract:Abstract The cytotoxic lectin Abrin shows more than 30 variant forms (R. Hegde, T. K. Maiti, and S. K. Podder, 1991, Anal. Biochem. 194, 101–109). The lectin B subunit as cause for variance in Abrins I and III was detected by a combination of one- and two-dimensional electrophoresis and Western blotting. Intriguingly, in Abrin I but not in Abrin III, association of a single A subunit with the variant B subunits shifts the holoprotein p I toward the alkaline side indicating that the subunit association involves neutralization of few negative charges. The B-subunit variants of Abrins I and III overlap in their p I , and the A-subunit association gives the holoproteins a distinctness on isoelectric focusing gel. The results were also confirmed by analyzing the pH titration curves. These differences in the subunit association pattern between Abrins I and III are in corroboration with the previously observed differences in the kinetics of protein synthesis inactivation and accessibility of the disulfide bridge to reducing agents in the presence or absence of putative receptor (R. Hegde, A. Karande, and S. K. Podder, 1993 Eur. J. Biochem. 215, 411–419). Further, the genetic origin of variance was confirmed by peptide mapping of the individual subunit variants. Considering a theoretical value of 0.1 to 0.2 p I /charge, a 15–17 charge difference could be predicted between the variants of two extreme p I s. The fact that the A subunits are not shared between the groups was taken to interpret that the protein synthesized as prepro form is processed posttranslationally and the processing takes place only after the disulfide bond formation between A and B subunits. The N-terminal 16 amino acids of A subunits of Abrins I and III showed 26% dissimilarity. The A subunits of Abrins I and III did not react with concanavalin A, indicating that the heterogeneity in the molecular weight is because of differential processing but not because of glycosylation.
-
The variants of the protein toxins Abrin and ricin. A useful guide to understanding the processing events in the toxin transport.
European journal of biochemistry, 1993Co-Authors: Ramesh Hegde, Anjali A Karande, S. K. PodderAbstract:Kinetic data on inhibition of protein synthesis in thymocyte by three Abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by k1-versus-concentration plots were Abrin II, III > ricin > Abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of Abrin I, II, III and ricin, respectively. To account for the observed differences in the dose-dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low-pH-induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that Abrin-I (4.13 × 105 M−1 s−1) association with putative receptor (4-methylumbelliferyl-α-D-galactoside) is nearly two times more often than Abrin III (2.6 × 105 M−1 s−1) at 20°C. Equillibrium binding constants of Abrin I and II to thymocyte at 37°C were 2.26 × 107 M−1 and 2.8 × 107 M−1 respectively. pH-induced structural alterations as studied by a parallel enhancement in 8-anilino-L-naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration-independent lag time (minimum lag of 41–42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose-dependent lag time is causally related to the proposed processing event. The rates of reduction of inter-subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of Abrin I is fourfold faster than that of Abrin II at pH 7.2. The rate of disulfide reduction in Abrin I could be decreased 1 I-fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose-dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.
-
purification and characterization of three toxins and two agglutinins from abrus precatorius seed by using lactamyl sepharose affinity chromatography
Analytical Biochemistry, 1991Co-Authors: Ramesh Hegde, Tapas K Maiti, S. K. PodderAbstract:Abstract Three toxins, Abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. Abrin-I did not bind on DEAE-Sephacel column chromatography and the bound Abrin-II, Abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are Abrin-I, 64,000; Abrin-II and Abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms−1 at a 0.63 n m concentration of each of Abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic Abrin-I and the highly toxic Abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.
