The Experts below are selected from a list of 87 Experts worldwide ranked by ideXlab platform

Varun P. Appanna - One of the best experts on this subject based on the ideXlab platform.

  • Deciphering metabolic networks by blue native polyacrylamide gel electrophoresis: A functional proteomic exploration
    EuPA Open Proteomics, 2015
    Co-Authors: Christopher Auger, Nishma D. Appanna, Azhar Alhasawi, Varun P. Appanna
    Abstract:

    Abstract Metabolism is the consortium of reactions within a cell which directs a variety of processes including energy synthesis, signalling and the behaviour of a biological system. Metabolic networks, and more specifically the activity of enzymes within them, provide an accurate status of how cellular information is being executed. The performance of these networks and their ability to siphon metabolites in a number of directions may be the difference between a healthy and diseased state. Blue native polyacrylamide gel electrophoresis (BN-PAGE), owing to its simplicity and wide-ranging applications, permits the inspection of these nodules. The separation of proteins and enzyme complexes in their native format enables the exploration of enzymatic activity in metabolic networks via in-gel assays. These are quick, specific, and amenable to further studies. This Electrophoretic Technology not only enables the visualization of enzymatic efficacy but reveals the crosstalk among enzymes and their interactions with other organellar partners.

  • A blue native polyacrylamide gel Electrophoretic Technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.
    Journal of Microbiological Methods, 2012
    Co-Authors: Christopher Auger, Varun P. Appanna, Joseph Lemire, Zachary Castonguay, Elchin Akbarov
    Abstract:

    As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective Electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This Electrophoretic Technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

  • A facile Electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases
    Electrophoresis, 2012
    Co-Authors: Christopher Auger, Varun P. Appanna, Zachary Castonguay, Sungwon Han
    Abstract:

    Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective Electrophoretic Technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.

Christopher Auger - One of the best experts on this subject based on the ideXlab platform.

  • Deciphering metabolic networks by blue native polyacrylamide gel electrophoresis: A functional proteomic exploration
    EuPA Open Proteomics, 2015
    Co-Authors: Christopher Auger, Nishma D. Appanna, Azhar Alhasawi, Varun P. Appanna
    Abstract:

    Abstract Metabolism is the consortium of reactions within a cell which directs a variety of processes including energy synthesis, signalling and the behaviour of a biological system. Metabolic networks, and more specifically the activity of enzymes within them, provide an accurate status of how cellular information is being executed. The performance of these networks and their ability to siphon metabolites in a number of directions may be the difference between a healthy and diseased state. Blue native polyacrylamide gel electrophoresis (BN-PAGE), owing to its simplicity and wide-ranging applications, permits the inspection of these nodules. The separation of proteins and enzyme complexes in their native format enables the exploration of enzymatic activity in metabolic networks via in-gel assays. These are quick, specific, and amenable to further studies. This Electrophoretic Technology not only enables the visualization of enzymatic efficacy but reveals the crosstalk among enzymes and their interactions with other organellar partners.

  • A blue native polyacrylamide gel Electrophoretic Technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.
    Journal of Microbiological Methods, 2012
    Co-Authors: Christopher Auger, Varun P. Appanna, Joseph Lemire, Zachary Castonguay, Elchin Akbarov
    Abstract:

    As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective Electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This Electrophoretic Technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

  • A facile Electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases
    Electrophoresis, 2012
    Co-Authors: Christopher Auger, Varun P. Appanna, Zachary Castonguay, Sungwon Han
    Abstract:

    Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective Electrophoretic Technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.

Frank N. Chang - One of the best experts on this subject based on the ideXlab platform.

  • G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer.
    Experimental and molecular pathology, 2011
    Co-Authors: George P. Tuszynski, Vicki L. Rothman, Xiaoyi Zheng, Mohammed Gutu, Xinmin Zhang, Frank N. Chang
    Abstract:

    Abstract An innovative “2-D high performance liquid electrophoresis” (2-D HPLE) Technology was used to identify serum biomarkers associated with the early stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed Electrophoretic Technology that separates 100s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins or their fragments (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of Stage 2 and Stage 3 breast cancers, but not in adjacent normal tissue as revealed by western blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE Technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all of 107 cases of archived ductal breast carcinoma tumor samples, while normal adjacent breast tissue from 12 cases of ductal carcinoma showed little or no staining. Additionally, all 10 cases of metastatic breast carcinoma present in lymph nodes were positive. Strong positive GASP-1 staining was observed in all tumor tissue including ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. Additionally, we observed a wide spectrum of enhanced staining of premalignant ductal epithelial cells present in benign ducts and those found in atypical ductal hyperplasia (ADH). These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for the development of breast cancer therapeutics.

  • Abstract 3183: The role of G-protein coupled receptor-associated sorting protein 1 (GASP-1) in breast cancer progression
    Clinical Research, 2011
    Co-Authors: George P. Tuszynski, Vicki L. Rothman, Mohammed Gutu, Frank N. Chang
    Abstract:

    An innovative “2-D High Performance Liquid Electrophoresis” (2-D HPLE) Technology was used to identify serum biomarkers associated with the earliest stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed Electrophoretic Technology that separates 1009s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that G-protein coupled receptor-associated sorting protein 1(GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of stage 2 and stage 3 breast cancer but not in adjacent normal tissue as revealed by Western Blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE Technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all 59 cases of archived ductal breast carcinoma tumor samples. When GASP-1 was silenced in MB-231 breast carcinoma, the cells grew more than 5 fold slower than corresponding vector controls. These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for development of breast cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2011-3183

George P. Tuszynski - One of the best experts on this subject based on the ideXlab platform.

  • G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer.
    Experimental and molecular pathology, 2011
    Co-Authors: George P. Tuszynski, Vicki L. Rothman, Xiaoyi Zheng, Mohammed Gutu, Xinmin Zhang, Frank N. Chang
    Abstract:

    Abstract An innovative “2-D high performance liquid electrophoresis” (2-D HPLE) Technology was used to identify serum biomarkers associated with the early stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed Electrophoretic Technology that separates 100s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins or their fragments (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of Stage 2 and Stage 3 breast cancers, but not in adjacent normal tissue as revealed by western blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE Technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all of 107 cases of archived ductal breast carcinoma tumor samples, while normal adjacent breast tissue from 12 cases of ductal carcinoma showed little or no staining. Additionally, all 10 cases of metastatic breast carcinoma present in lymph nodes were positive. Strong positive GASP-1 staining was observed in all tumor tissue including ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. Additionally, we observed a wide spectrum of enhanced staining of premalignant ductal epithelial cells present in benign ducts and those found in atypical ductal hyperplasia (ADH). These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for the development of breast cancer therapeutics.

  • Abstract 3183: The role of G-protein coupled receptor-associated sorting protein 1 (GASP-1) in breast cancer progression
    Clinical Research, 2011
    Co-Authors: George P. Tuszynski, Vicki L. Rothman, Mohammed Gutu, Frank N. Chang
    Abstract:

    An innovative “2-D High Performance Liquid Electrophoresis” (2-D HPLE) Technology was used to identify serum biomarkers associated with the earliest stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed Electrophoretic Technology that separates 1009s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that G-protein coupled receptor-associated sorting protein 1(GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of stage 2 and stage 3 breast cancer but not in adjacent normal tissue as revealed by Western Blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE Technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all 59 cases of archived ductal breast carcinoma tumor samples. When GASP-1 was silenced in MB-231 breast carcinoma, the cells grew more than 5 fold slower than corresponding vector controls. These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for development of breast cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2011-3183

Zachary Castonguay - One of the best experts on this subject based on the ideXlab platform.

  • A blue native polyacrylamide gel Electrophoretic Technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.
    Journal of Microbiological Methods, 2012
    Co-Authors: Christopher Auger, Varun P. Appanna, Joseph Lemire, Zachary Castonguay, Elchin Akbarov
    Abstract:

    As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective Electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This Electrophoretic Technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

  • A facile Electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases
    Electrophoresis, 2012
    Co-Authors: Christopher Auger, Varun P. Appanna, Zachary Castonguay, Sungwon Han
    Abstract:

    Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective Electrophoretic Technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.