- Email: [email protected]
A novel beta-lactamase from subsurface sediments
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
N2 Identification of three newly biomolecules (CC3-SPase, CCSV-MPase and CC2-PLA2) isolated from Cerastes cerastes venom: Proteomic analysis Cherifi Fatah 1,2 1
University of Sciences and Technology Houari Boumediene Bab Ezzouar, Department of Cellular and Molecular Biology, Algiers, Algeria 2 Pasteur Institute of Algiers, Laboratory of Researches and Development on Venoms, Algiers, Algeria E-mail address: cherifi[email protected]
Viperidae venom is a rich source of bioactive molecules. Proteomic analysis was used to identify molecules from whole venom. Three biomolecules newly isolated from Cerastes cerastes venom have been analyzed by Nano-liquid Chromatography and Mass Spectrometry. After reduction, alkylation and double hydrolysis of the molecules by Lysine-C endopeptidase and trypsin, LCMALDI-MS/MS was performed. The proteins were identified by mining the database using the NCBInr protein. Obtained results revealed the presence of another phospholipase activity (CC2PLA2) which shares 51% of homology sequence with the previous purified PLA2 from the same venom. CC2-PLA2 exhibits antiplatelet activity leading to a coagulation blockade. CCSV-MPase, a non-haemorrhagic Zn2+ -metalloproteinase, reduced significantly the plasmatic fibrinogen level and hydrolyzed only its B( chain. However, CC3-SPase (a serine protease) could be an ( ( fibrinogenase triggering human platelet aggregation. In the presence of human plasma deficient in Factor II, CC3-SPase revealed a blood-clotting activity comparable to that obtained with normal plasma. However, the clot so obtained was soluble indicating that CC3-SPase does not activate factor XIII. These results imply that CC3-SPase could repair haemostatic abnormalities and may replace some factors in deficient pathology. doi:10.1016/j.copbio.2011.05.399
N3 Expression profiling of apoptotic proteins in human breast cancer cells treated with AT-101 by protein array technology Harika Atmaca 1 , Burcak Karaca 2 , Asli Kisim 1 , Selim Uzunoglu 1 , Ruchan Uslu 2 1
S123
vivin, XIAP, and cIAP2) are reduced in MCF-7 cells. However, the expression of proapoptotic proteins (Bax, Fas, FADD, HSP60, and HSP70) are induced and the antiapoptotic proteins (cIAP1, cIAP2, clusterin, and pro-caspase3) are reduced in AT-101 treated MDAMB-231 cells. This kind of omic analysis of the apoptotic response may yield insights into the action mechanism of potential anticancer agents such as AT-101 and allow rational design of more effective treatment strategies for breast cancer. doi:10.1016/j.copbio.2011.05.400
N4 Horizontal gene transfer (HGT): A driving force for resistance dissemination in gut microbiota Ibrahim Farouk Farag 1 , Magda Mahmoud Hagag 2 1
Biotechnology Program, American University in Cairo (AUC), Egypt Biochemistry Department, Faculty of Medicine, Ain Shams University, Egypt 2
E-mail address: [email protected] (M.M. Hagag) Microbes are continuously exposed to different environmental stresses promoting it to exhibit mechanisms neutralizing these threats. Among these mechanisms, HGT plays pivotal roles in processing genes movement utilizing the power of different mobile elements which exist in microbes’ chromosomal and extra-chromosomal spaces. To estimate HGT roles in enhancing the gut-dwelling flora resistome, comparative analyses using SEED database were conducted on metagenomic data of six different gut microbiomes. Two of which belong to lean and obese mice and the other four were done on human adult subjects. Interestingly, results revealed high abundances of phages and phage-related genes especially in human gut, followed by transposable elements and insignificant presence of plasmids. Additionally, conjugative transposons especially those belonging to Bacteroidales were highly represented in most of metagenomes tested unless it was questionably absent in obese mice microbiome. These elements enriched the gut resistance gene pools which were represented by several classes, including beta-lactamases, methicillin and fluoroquinolones resistance genes. Interestingly, genes encoding heavy metals’ resistances exhibited remarkable differences between human and mice microbiomes as it was completely absent in the later. We are hoping that this study may shed some of the light on the relations between HGT and antibiotic resistance in gut environments.
Department of Biology, School of Science and Arts, Celal Bayar University, Manisa, Turkey 2 Tulay Aktas Oncology Hospital, School of Medicine, Ege University, Izmir, Turkey
doi:10.1016/j.copbio.2011.05.401
E-mail address: [email protected] (H. Atmaca)
A novel beta-lactamase from subsurface sediments
Protein expression profiling with protein array technology allows simultaneous analysis of the protein expression pattern of a large number of proteins. AT-101 is an (−)-enantiomer of gossypol and it was reported to have potent antiproliferative effects. To determine the multiple expression pattern of apoptotic proteins in human breast cancer cell lines (MCF-7 and MDA-MB-231) treated with AT-101 agent. XTT cell proliferation kit and Cell Death Detection Elisa Plus Kit (Roche) was used for measuring cytotoxicity and apoptosis. Human Apoptosis Protein Array (R&D Systems) which consists of 35 apoptosis related proteins was used to assess the omic protein expression pattern. The expression of proapoptotic proteins (TRAIL R1 and R2, caspase 3, Bad, and cytochrome c) are induced, while the expression of antiapoptotic proteins (Bcl-2, sur-
Nurcan Vardar 1 , Gonul Vardar Schara 1 , Gonul Vardar Schara 2
N5
1
Department of Genetics and Bioengineering, Fatih University, Istanbul, Turkey 2 Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, USA E-mail address: [email protected] (N. Vardar) The emergence and spread of antibiotic resistance among environmental bacteria may become a global threat in our near future. Knowledge on novel antibiotic resistance genes from natural environments is necessary in order to minimize this problem. Metagenomics approach paves a pathway to search out novel genes
S124
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
by enabling us to access the unrecognized microbial diversity. The aim of this study was to attain more insight into the frequency and diversity of antibiotic resistance genes present in terrestrial subsurface environment which received relatively little attention, using metagenomic tools. Pre-enriched metagenomic libraries were constructed from soil/rock samples that were collected from 24 m below land surface in Hawaii and screened for resistance to antibiotics including kanamycin, ampicillin, and neomycin. All resistant clones were evaluated by restriction digest, sequenced by primer walking, and the genes responsible were identified by subcloning. A novel class A beta-lactamase with 307 amino acids that is most similar to the penicillinase from Bacillus licheniformis was discovered and its minimum inhibitory concentration values were measured against several beta-lactam antibiotics. This study extends the knowledge on resistance to antimicrobials, which may help the efforts to slow down their spread and prevent the revival of infectious diseases.
Although they are very effective, several bacteria are shown to have resistance against those antibiotics. Our aim in this study was to develop a different method to control quinolone resistant bacteria. Quinolone resistant Salmonella typhimurium TA1535 was prepared by applying different concentrations of ciprofloxacin. Three Bacillus species including B. subtilis, B. megaterium, B. cereus were overcultured in TSB for 1w.Then extracellular proteins were extracted from the supernatants of centrifugated bacterial cells. Intracellular proteins from bacterial strains tested were extracted by using Easylyse Protein Extraction Kit. The inhibition activity of extracellular and intracellular proteins was determined based on disc diffusion assay. The intracellular proteins of Bacillus subtilis strains and also Bacillus megaterium was found effective on ciprofloxacin resistant Salmonella typhimurium TA1535. This result suggested that secondary metabolites in Bacillus strains can be used as potential antibacterial agent for quinolone resistant bacteria. doi:10.1016/j.copbio.2011.05.404
doi:10.1016/j.copbio.2011.05.402
O3
Pharmaceutical Biotechnology Section O1 Significant reduction of IgG aggregates from therapeutic monoclonal antibodies Nizar Mohammad Abuharfeil 1,2 , Barakat Mohammad Shabsoug 1,2 1
Dept. Biotechnology, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan 2 Dept. Chemistry, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan E-mail address: [email protected] (N.M. Abuharfeil) The therapeutic monoclonal antibody rituximab like all IgG monoclonal antibodies show self aggregates. These aggregates may cause reduction in antibody efficacy, elicit an unfavorable immunological response and consequently affect the specification of the product. The aim is to remove IgG aggregates as much as possible. IgG aggregation was increased by incubation of the rituximab antibodies at 50 ◦ C for half an hour. Fifty mg/ml of the monoclonal antibody was applied to hydroxyl-apatite chromatography using 5% polyethylene glycol in 10 mM PBS buffer, pH 7.2. The eluted IgG sample was dissolved in 0.15 M arginine hydrochloride in 10 mM PBS buffer, pH 7.2. Assay of aggregates was performed by size exclusion chromatography-HPLC and ELISA. The amount of IgG aggregates was reduced from 16.3% to 0.1% after hydroxyl apatite chromatography. The addition of arginine hydrochloride reduced further the aggregate to 0.01%. This result might be of useful value for the preparation of aggregate-free therapeutic monoclonal antibodies or IVIG in pharmaceutical industries. doi:10.1016/j.copbio.2011.05.403
O2 Antibacterial effect of secondary metabolites of Bacillus species against quinolone resistant bacteria Dilsad Yurdakul, Fikrettin Sahin Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul, Turkey E-mail address: [email protected] (D. Yurdakul) Quinolones are antibiotics that inhibit the bacterial DNA gyrase enzyme thereby inhibiting the synthesis of DNA and protein.
Investigating of interactions between statin-based cholesterol lowering drugs with p-glycoprotein membrane protein by molecular modeling Deniz Karasu 1 , Fatma Nese Kok 1,2 , Cenk Selcuki 3 1
Molecular Biology-Genetics and Biotechnology Program, MOBGAM, Istanbul Technical University, Istanbul, Turkey 2 Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul, Turkey 3 Department of Biochemistry, Faculty of Science, Ege University, Izmir, Turkey E-mail address: [email protected] (D. Karasu) The interaction of P-glycoprotein (P-gp) with lactone form of cholesterol lowering atorvastatin and linear peptides including ALLM (N-acetyl-leu-leu-met-al) and AFMRF (N-acetyl-phe-metarg-phe-al) were investigated by using molecular dynamics simulation. P-gp is a multidrug resistance protein which expels the toxic compounds out of the cell and in this way protects the cell from the harmful effect of these compounds. P-gp was integrated into DMPC lipid bilayer and solvated to investigate these interactions. Then MgATP molecules, which supply the required energy for transportation, were docked into the nucleotide binding domains of the protein. After the preparation of the P-gp integrated lipid membrane system, simulations were run for each substrate. While no interaction was detected between the linear peptides and Pgp, lactone form of atorvastatin was found to have an interaction with the protein, leading to an asymmetrical closure of nucleotide binding domains. When the ability of P-gp to transport multiple substrates were taken into account, it could be said that single small peptides could not trigger the protein activity needed for transportation. doi:10.1016/j.copbio.2011.05.405
N2 Identification of three newly biomolecules (CC3-SPase, CCSV-MPase and CC2-PLA2) isolated from Cerastes cerastes venom: Proteomic analysis Cherifi Fatah 1,2 1
University of Sciences and Technology Houari Boumediene Bab Ezzouar, Department of Cellular and Molecular Biology, Algiers, Algeria 2 Pasteur Institute of Algiers, Laboratory of Researches and Development on Venoms, Algiers, Algeria E-mail address: cherifi[email protected]
Viperidae venom is a rich source of bioactive molecules. Proteomic analysis was used to identify molecules from whole venom. Three biomolecules newly isolated from Cerastes cerastes venom have been analyzed by Nano-liquid Chromatography and Mass Spectrometry. After reduction, alkylation and double hydrolysis of the molecules by Lysine-C endopeptidase and trypsin, LCMALDI-MS/MS was performed. The proteins were identified by mining the database using the NCBInr protein. Obtained results revealed the presence of another phospholipase activity (CC2PLA2) which shares 51% of homology sequence with the previous purified PLA2 from the same venom. CC2-PLA2 exhibits antiplatelet activity leading to a coagulation blockade. CCSV-MPase, a non-haemorrhagic Zn2+ -metalloproteinase, reduced significantly the plasmatic fibrinogen level and hydrolyzed only its B( chain. However, CC3-SPase (a serine protease) could be an ( ( fibrinogenase triggering human platelet aggregation. In the presence of human plasma deficient in Factor II, CC3-SPase revealed a blood-clotting activity comparable to that obtained with normal plasma. However, the clot so obtained was soluble indicating that CC3-SPase does not activate factor XIII. These results imply that CC3-SPase could repair haemostatic abnormalities and may replace some factors in deficient pathology. doi:10.1016/j.copbio.2011.05.399
N3 Expression profiling of apoptotic proteins in human breast cancer cells treated with AT-101 by protein array technology Harika Atmaca 1 , Burcak Karaca 2 , Asli Kisim 1 , Selim Uzunoglu 1 , Ruchan Uslu 2 1
S123
vivin, XIAP, and cIAP2) are reduced in MCF-7 cells. However, the expression of proapoptotic proteins (Bax, Fas, FADD, HSP60, and HSP70) are induced and the antiapoptotic proteins (cIAP1, cIAP2, clusterin, and pro-caspase3) are reduced in AT-101 treated MDAMB-231 cells. This kind of omic analysis of the apoptotic response may yield insights into the action mechanism of potential anticancer agents such as AT-101 and allow rational design of more effective treatment strategies for breast cancer. doi:10.1016/j.copbio.2011.05.400
N4 Horizontal gene transfer (HGT): A driving force for resistance dissemination in gut microbiota Ibrahim Farouk Farag 1 , Magda Mahmoud Hagag 2 1
Biotechnology Program, American University in Cairo (AUC), Egypt Biochemistry Department, Faculty of Medicine, Ain Shams University, Egypt 2
E-mail address: [email protected] (M.M. Hagag) Microbes are continuously exposed to different environmental stresses promoting it to exhibit mechanisms neutralizing these threats. Among these mechanisms, HGT plays pivotal roles in processing genes movement utilizing the power of different mobile elements which exist in microbes’ chromosomal and extra-chromosomal spaces. To estimate HGT roles in enhancing the gut-dwelling flora resistome, comparative analyses using SEED database were conducted on metagenomic data of six different gut microbiomes. Two of which belong to lean and obese mice and the other four were done on human adult subjects. Interestingly, results revealed high abundances of phages and phage-related genes especially in human gut, followed by transposable elements and insignificant presence of plasmids. Additionally, conjugative transposons especially those belonging to Bacteroidales were highly represented in most of metagenomes tested unless it was questionably absent in obese mice microbiome. These elements enriched the gut resistance gene pools which were represented by several classes, including beta-lactamases, methicillin and fluoroquinolones resistance genes. Interestingly, genes encoding heavy metals’ resistances exhibited remarkable differences between human and mice microbiomes as it was completely absent in the later. We are hoping that this study may shed some of the light on the relations between HGT and antibiotic resistance in gut environments.
Department of Biology, School of Science and Arts, Celal Bayar University, Manisa, Turkey 2 Tulay Aktas Oncology Hospital, School of Medicine, Ege University, Izmir, Turkey
doi:10.1016/j.copbio.2011.05.401
E-mail address: [email protected] (H. Atmaca)
A novel beta-lactamase from subsurface sediments
Protein expression profiling with protein array technology allows simultaneous analysis of the protein expression pattern of a large number of proteins. AT-101 is an (−)-enantiomer of gossypol and it was reported to have potent antiproliferative effects. To determine the multiple expression pattern of apoptotic proteins in human breast cancer cell lines (MCF-7 and MDA-MB-231) treated with AT-101 agent. XTT cell proliferation kit and Cell Death Detection Elisa Plus Kit (Roche) was used for measuring cytotoxicity and apoptosis. Human Apoptosis Protein Array (R&D Systems) which consists of 35 apoptosis related proteins was used to assess the omic protein expression pattern. The expression of proapoptotic proteins (TRAIL R1 and R2, caspase 3, Bad, and cytochrome c) are induced, while the expression of antiapoptotic proteins (Bcl-2, sur-
Nurcan Vardar 1 , Gonul Vardar Schara 1 , Gonul Vardar Schara 2
N5
1
Department of Genetics and Bioengineering, Fatih University, Istanbul, Turkey 2 Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, USA E-mail address: [email protected] (N. Vardar) The emergence and spread of antibiotic resistance among environmental bacteria may become a global threat in our near future. Knowledge on novel antibiotic resistance genes from natural environments is necessary in order to minimize this problem. Metagenomics approach paves a pathway to search out novel genes
S124
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
by enabling us to access the unrecognized microbial diversity. The aim of this study was to attain more insight into the frequency and diversity of antibiotic resistance genes present in terrestrial subsurface environment which received relatively little attention, using metagenomic tools. Pre-enriched metagenomic libraries were constructed from soil/rock samples that were collected from 24 m below land surface in Hawaii and screened for resistance to antibiotics including kanamycin, ampicillin, and neomycin. All resistant clones were evaluated by restriction digest, sequenced by primer walking, and the genes responsible were identified by subcloning. A novel class A beta-lactamase with 307 amino acids that is most similar to the penicillinase from Bacillus licheniformis was discovered and its minimum inhibitory concentration values were measured against several beta-lactam antibiotics. This study extends the knowledge on resistance to antimicrobials, which may help the efforts to slow down their spread and prevent the revival of infectious diseases.
Although they are very effective, several bacteria are shown to have resistance against those antibiotics. Our aim in this study was to develop a different method to control quinolone resistant bacteria. Quinolone resistant Salmonella typhimurium TA1535 was prepared by applying different concentrations of ciprofloxacin. Three Bacillus species including B. subtilis, B. megaterium, B. cereus were overcultured in TSB for 1w.Then extracellular proteins were extracted from the supernatants of centrifugated bacterial cells. Intracellular proteins from bacterial strains tested were extracted by using Easylyse Protein Extraction Kit. The inhibition activity of extracellular and intracellular proteins was determined based on disc diffusion assay. The intracellular proteins of Bacillus subtilis strains and also Bacillus megaterium was found effective on ciprofloxacin resistant Salmonella typhimurium TA1535. This result suggested that secondary metabolites in Bacillus strains can be used as potential antibacterial agent for quinolone resistant bacteria. doi:10.1016/j.copbio.2011.05.404
doi:10.1016/j.copbio.2011.05.402
O3
Pharmaceutical Biotechnology Section O1 Significant reduction of IgG aggregates from therapeutic monoclonal antibodies Nizar Mohammad Abuharfeil 1,2 , Barakat Mohammad Shabsoug 1,2 1
Dept. Biotechnology, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan 2 Dept. Chemistry, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan E-mail address: [email protected] (N.M. Abuharfeil) The therapeutic monoclonal antibody rituximab like all IgG monoclonal antibodies show self aggregates. These aggregates may cause reduction in antibody efficacy, elicit an unfavorable immunological response and consequently affect the specification of the product. The aim is to remove IgG aggregates as much as possible. IgG aggregation was increased by incubation of the rituximab antibodies at 50 ◦ C for half an hour. Fifty mg/ml of the monoclonal antibody was applied to hydroxyl-apatite chromatography using 5% polyethylene glycol in 10 mM PBS buffer, pH 7.2. The eluted IgG sample was dissolved in 0.15 M arginine hydrochloride in 10 mM PBS buffer, pH 7.2. Assay of aggregates was performed by size exclusion chromatography-HPLC and ELISA. The amount of IgG aggregates was reduced from 16.3% to 0.1% after hydroxyl apatite chromatography. The addition of arginine hydrochloride reduced further the aggregate to 0.01%. This result might be of useful value for the preparation of aggregate-free therapeutic monoclonal antibodies or IVIG in pharmaceutical industries. doi:10.1016/j.copbio.2011.05.403
O2 Antibacterial effect of secondary metabolites of Bacillus species against quinolone resistant bacteria Dilsad Yurdakul, Fikrettin Sahin Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul, Turkey E-mail address: [email protected] (D. Yurdakul) Quinolones are antibiotics that inhibit the bacterial DNA gyrase enzyme thereby inhibiting the synthesis of DNA and protein.
Investigating of interactions between statin-based cholesterol lowering drugs with p-glycoprotein membrane protein by molecular modeling Deniz Karasu 1 , Fatma Nese Kok 1,2 , Cenk Selcuki 3 1
Molecular Biology-Genetics and Biotechnology Program, MOBGAM, Istanbul Technical University, Istanbul, Turkey 2 Molecular Biology and Genetics Department, Istanbul Technical University, Istanbul, Turkey 3 Department of Biochemistry, Faculty of Science, Ege University, Izmir, Turkey E-mail address: [email protected] (D. Karasu) The interaction of P-glycoprotein (P-gp) with lactone form of cholesterol lowering atorvastatin and linear peptides including ALLM (N-acetyl-leu-leu-met-al) and AFMRF (N-acetyl-phe-metarg-phe-al) were investigated by using molecular dynamics simulation. P-gp is a multidrug resistance protein which expels the toxic compounds out of the cell and in this way protects the cell from the harmful effect of these compounds. P-gp was integrated into DMPC lipid bilayer and solvated to investigate these interactions. Then MgATP molecules, which supply the required energy for transportation, were docked into the nucleotide binding domains of the protein. After the preparation of the P-gp integrated lipid membrane system, simulations were run for each substrate. While no interaction was detected between the linear peptides and Pgp, lactone form of atorvastatin was found to have an interaction with the protein, leading to an asymmetrical closure of nucleotide binding domains. When the ability of P-gp to transport multiple substrates were taken into account, it could be said that single small peptides could not trigger the protein activity needed for transportation. doi:10.1016/j.copbio.2011.05.405