- Email: [email protected]
Purification and immunogenic characterization of anti-LPS rabbit antibodies produced against Salmonella Typhimurium extracted lipopolysaccharide
S356
Abstracts
strated. In the present study, acidic fibrillation of hen–egg weight lysozyme (HEWL) was analyzed in the presence of crude and carboxyl-functionalized nanotubes. Methods: Multi-walled CNTs were acid oxidized to form carboxylic acid group on the surface of the CNTs. IR-spectroscopy was used to verify the CNT functionalization. A sample solution of 2 mg/ml HEWL in acidic water with salts was prepared. Nanotubes at two final concentrations were added to HEWL solution and were incubated at 55 °C at different times. Congo red binding assay and Far-UV CD spectroscopy were used to characterize amyloid fibrillation. Results and discussion: Results from Congo red absorption spectroscopy demonstrated the formation of fibrillar amyloid structures at 72 h after the initiation of HEWL incubation. In addition, CD spectra of HEWL exhibited a characteristic pattern of β-sheet conformation. In exploring if CNT exerted a stimulatory action on the formation of HEWL fibrils, we monitored the changes of Congo red binding and CD spectra in amyloidogenic conditions. Our observations indicated that crude CNT could outstandingly enhance amyloid fibril formation while CNT in carboxylated form had no significant effect on fibrillation process. Conclusion: It is concluded that introduction of polar groups on the hydrophobic surfaces may play an important inhibitory role on surface-assisted nucleation. Keywords: Carbon nanotube, Protein folding, Fibrillation, Carboxylation doi:10.1016/j.clinbiochem.2011.08.895
Poster — [A-10-225-1] Cytotoxic potential of five Iranian medicinal plants by Artemia urmiana Mehdi Ghahremanifar, Ali Mirzaei, Maasoomeh Kazemi Yasouj Medical Sciences University, Medical College, Yasouj, Iran E-mail addresses: [email protected] (M. Ghahremanifar), [email protected] (A. Mirzaei), [email protected] (M. Kazemi) Introduction: Many plant products have strong anticancer activity against cancer cells (human and rodents). Phytochemical compounds in plants causing to strong antioxidant activity that may prevent from some cancers or some other diseases through inhibition of free radicals. Thus, antioxidant-rich plants can cause health and prevent from growing of cancer. One of the most reliable tests is the brine shrimp lethality test (BLST) that is simple procedure for screening of plant toxicity and bioactive compounds having antitumor activity. Material and methods: In the present study, aqueous extracts of Mentha logifolia, Achillea mellefolium, Mentha piperita, Matricaria recutita and Teucrium polium medicinal plants were screened for their cytotoxicity using brine shrimp lethality test. Five concentrations (25, 50, 75, 100, and 150 ppm) of each extract were applied in the bioassay and the LC50 determined. Results: The least LC50 value of 32 was obtained for hexane extract and the highest value of 13.87 was obtained for aqueous extract using the Finneys probit analysis which indicates high lethality of the extracts and presence of potent cytotoxic compounds in the plant extracts. Out of the 5 plants tested, M. recutita exhibited potent brine shrimp lethality with LC50 32 μg. M. logifolia, A. mellefolium, T. polium and M. piperita have also showed moderate cytotoxicity with LC50 111, 160, 172 and 247 μg respectively. Conclusion: The present study supports that brine shrimp bioassay is simple reliable and convenient method for evalution of bioactivity of medicinal plants and lends support for their use in traditional medicine.
Keywords: Artemia urmiana, Brine shrimp lethality test, Medicinal plants, Cytotoxicity doi:10.1016/j.clinbiochem.2011.08.896
Poster — [A-10-265-1] Beneficial effects of allicin on lead induced tissue enzyme alterations in common carp (Cyprinus carpio) Hasan Baghshani, Davar Shahsavani Ferdowsi University of Mashhad, Mashhad, Iran E-mail addresses: [email protected] (H. Baghshani), [email protected] (D. Shahsavani) Introduction: In toxicological studies, the alterations in the enzymatic activities directly reflect the metabolic disturbances and cell damage in specific organs. The aim of the current study was to determine the effects of lead exposure on tissue enzyme activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in selected tissues of common carp and also to evaluate the beneficial effects of allicin, the main active constituent of garlic, on modulating enzyme alterations. Methods: Common carp (Cyprinus carpio; total, n = 60) were divided randomly into three groups of 20 each. Group I fish were reared in normal freshwater and served as the control. Group II fish were exposed to lead acetate (7.0 mg L−1) for 10 days. Group III fish were exposed to lead acetate (7.0 mg L−1) for 10 days and also received dietary allicin (10 mg/kg body weight, daily) during lead acetate treatment. At the end of each exposure, enzyme activities in prepared tissue homogenates were determined using colorimetric methods. Results: Lead exposure caused a significant elevation of AST, ALT and LDH in the liver, as compared to control group (P < 0.05). The AST, ALT, ALP and LDH levels in kidney homogenates were considerably increased following lead exposure, although these increases were only significant for ALT and ALP values. Significant increases of brain ALT and gill ALP activities were also observed following lead exposure. However, alterations in the activities of other measured enzymes in both brain and gill of experimental groups were not significant. Conclusion: Allicin supplementation was effective in attenuating the increase in Pb-induced tissue enzyme activities in the most organs; therefore it might have some protective and therapeutic effects on lead poisoning. However, much more extensive biochemical and toxicological research are needed for better understanding of the molecular basis of lead induced tissue enzyme alterations and ameliorative properties of allicin. Keywords: Lead poisoning, Allicin, Tissue enzyme activity, Common carp doi:10.1016/j.clinbiochem.2011.08.897
Poster — [A-10-274-1] Purification and immunogenic characterization of anti-LPS rabbit antibodies produced against Salmonella Typhimurium extracted lipopolysaccharide Ebrahimi Zadeh Waleada, Seyedlatif Mousavi Gargarib, Masoumeh Rajabi Bazlb, Mohammad Mohammadib a Shahed University, Qom, Iran b Tehran-qum Express Way, Shahed University, Iran E-mail addresses: [email protected] (E.Z. Walead), [email protected] (S.M. Gargari),
Abstracts
[email protected] (M. Rajabi Bazl), [email protected] (M. Mohammadi) Introduction: Salmonellae are anaerobic bacteria of the family Enterobacteriaceae. Salmonella enterica serovar Typhimurium is among the most common Salmonella serovars causing infections in human, livestock and poultry. O antigen (Lipopolysaccharide) is the dominant antigen, giving the bacteria ability to survive in intracellular conditions. In this study the S. typhimurium was neutralized with IgG purified from immunized rabbit with LPS and then used to challenge BALB/c mice. Methods: The O antigen from S. typhimurium was extracted with phenol-chloroform petroleum ether and analyzed with Tricine-SDSPAGE and LPS-Silver stain methods. Rabbits were immunized by LPS. Serum was isolated and antibody titer was assayed by ELISA. Antibody is extracted by ammonium sulfate precipitation followed by purification with DEAE cellulose chromatography. Attachment ability of antibody to LPS and S. typhimurium was analyzed by ELISA. S. typhimurium was incubated with antibody for 1 h at 37 °C and then gavaged into BALB/c female mice stomach. Control mice were only given same amount of bacterium. Results: Extracted LPS showed similar pattern compared to standard LPS in Tricine-SDS-PAGE. Antibody showed great affinity to extracted LPS and S. typhimurium. Compared to control mice, when the bacteria were treated with antibody the CFU for LD50 was significantly increased. Conclusion: Using bacterium as an antigen in the ELISA test, we showed that the affinity of antibody to S. typhimurium bacterium did not reduced compared to LPS. Neutralization test showed that produced antibody is able to attach to the bacterium and inhibit its colonization in in vivo experiment. Keywords: Salmonella Typhimurium, LPS, Antibody, LD50, BALB/c mice doi:10.1016/j.clinbiochem.2011.08.898
Poster — [A-10-275-1] Heavy chain camelied-antibodies (VHH) passively protect BALB/c mice against BoNT serotype E in neutralization test Hamid Bakherada, Latif Mousavi Gargaria, Masoumeh Rajabi Bazlb, Mohammad Mohammadic, Hamed Zarea, Leila Safaeea a Biology Department, Basic Science Faculty, Shahed University, Tehran, Iran b Biochemical Department, Medicine Faculty, Shahid Beheshti University, Tehran, Iran c Biology Department, Basic Science Faculty, Shahid Chamran University, Ahvaz, Iran E-mail addresses: [email protected] (H. Bakherad), [email protected] (L.M. Gargari), [email protected] (M. Rajabi Bazl), [email protected] (M. Mohammadi), [email protected] (H. Zare), [email protected] (L. Safaee) Introduction: Food intoxications caused by botulinium neurotoxins (BoNTs) are the most lethal poisoning known in the world. Thus the preventive and treatment measure are necessary particularly in the developing countries. In this study we investigated in vivo neutralization of BoNTE with Heavy Chain Camelied-Antibodies (VHH). Methods: VHH panning was done after library construction of the VHH gene amplified by nested PCR. High affinity colonies were cultured to produce VHH antibody. The recombinant VHH was purified by affinity chromatography using Ni-Nta Column. The LD50 for BoNTE was estimated. In vivo neutralization assay was done by mixing different concentrations of toxin with antibody and incubation for 1 h at 37 °C. The mixture was brought to 200 μl with PBS and injected to
S357
mice intraperitoneasly. The survival of mice was monitored after 24 h. Results: LD50 of the purified BoNTE toxin in our experiment was estimated to be around 2 ng per mouse. Results from neutralization tests for the VHH antibody showed a 100% survival of mice injected with a lethal dose of as high as 8 LD50 of BoNTE toxin (16 ng per mouse). Conclusions: The effect of the valency of the VHH antibody fragments on affinity and neutralizing capacity was the major goal of this study. The data obtained from our experiments highlight the realistic potential of novel recombinant antibody fragments to be used as a threupathic agent in order to reduce the lethality caused by BoNTE toxin. Keywords: BoNTE, VHH antibody, Neutralization
doi:10.1016/j.clinbiochem.2011.08.899
Poster — [A-10-323-1] Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species Gholamreza Karimia, Javad Behravanb, Fateme Mosaffab, Leila Hosseinzadehc, Bahrami Gholamrezac a Mashhad University of Medical Sciences, Faculty of Pharmacy, Mashhad, Iran b Pharmacy School, Mashhad, Iran c Pharmacy School, Kermanshah, Iran E-mail addresses: [email protected] (G. Karimi), [email protected] (J. Behravan), [email protected] (F. Mosaffa), [email protected] (L. Hosseinzadeh) Introduction: Doxorubicin (DOX) is a widely used chemotherapy agent. The major adverse effect of DOX treatment in cancer patients is the onset of cardiomyopathy and heart failure. Reactive oxygen species (ROS) are proposed to be responsible for DOX cardiotoxicity. Curcumin a natural compound extracted from Curcuma longa L. is known for its antioxidant properties. Therefore, we evaluated the effects of curcumin on apoptosis induced by DOX in cardiac muscle cells. Materials and methods: Detection of apoptosis was performed by Annexin V-FITC apoptosis detection kit with flow cytometry. Quantitative specific RNA expression was performed in one step with CYBR-green invitrogen kit. Intracellular ROS levels were examined using DCF-DA. Results: Preteatment with curcumin significantly increased DOXinduced apoptosis of cardiac muscle cells through down regulation of Bcl2, up-regulation of caspase-8 and caspase-9. The Bax/Bcl-2 ratio increased significantly after 1 h pretreatment with curcumin. Also curcumin increased ROS generation by DOX. In response to excess DOX, NF-κB was activated. However, curcumin was able to inhibit NF-κB activation. Conclusion: Our results indicated that pretreatment with nontoxic concentrations of curcumin sensitized H9c2 cells to DOX-mediated apoptosis by generation of ROS. Keywords: Doxorubicin, Curcumin, Apoptosis
doi:10.1016/j.clinbiochem.2011.08.900
Poster — [A-10-417-1] In vitro quantitative genotoxic analysis of two dentin bonding agents on primary human dental pulp cells Mohammad Ehsan Zangeneh, Glenn Oyong Manila, Philippines E-mail addresses: [email protected] (M.E. Zangeneh), [email protected] (G. Oyong)
Abstracts
strated. In the present study, acidic fibrillation of hen–egg weight lysozyme (HEWL) was analyzed in the presence of crude and carboxyl-functionalized nanotubes. Methods: Multi-walled CNTs were acid oxidized to form carboxylic acid group on the surface of the CNTs. IR-spectroscopy was used to verify the CNT functionalization. A sample solution of 2 mg/ml HEWL in acidic water with salts was prepared. Nanotubes at two final concentrations were added to HEWL solution and were incubated at 55 °C at different times. Congo red binding assay and Far-UV CD spectroscopy were used to characterize amyloid fibrillation. Results and discussion: Results from Congo red absorption spectroscopy demonstrated the formation of fibrillar amyloid structures at 72 h after the initiation of HEWL incubation. In addition, CD spectra of HEWL exhibited a characteristic pattern of β-sheet conformation. In exploring if CNT exerted a stimulatory action on the formation of HEWL fibrils, we monitored the changes of Congo red binding and CD spectra in amyloidogenic conditions. Our observations indicated that crude CNT could outstandingly enhance amyloid fibril formation while CNT in carboxylated form had no significant effect on fibrillation process. Conclusion: It is concluded that introduction of polar groups on the hydrophobic surfaces may play an important inhibitory role on surface-assisted nucleation. Keywords: Carbon nanotube, Protein folding, Fibrillation, Carboxylation doi:10.1016/j.clinbiochem.2011.08.895
Poster — [A-10-225-1] Cytotoxic potential of five Iranian medicinal plants by Artemia urmiana Mehdi Ghahremanifar, Ali Mirzaei, Maasoomeh Kazemi Yasouj Medical Sciences University, Medical College, Yasouj, Iran E-mail addresses: [email protected] (M. Ghahremanifar), [email protected] (A. Mirzaei), [email protected] (M. Kazemi) Introduction: Many plant products have strong anticancer activity against cancer cells (human and rodents). Phytochemical compounds in plants causing to strong antioxidant activity that may prevent from some cancers or some other diseases through inhibition of free radicals. Thus, antioxidant-rich plants can cause health and prevent from growing of cancer. One of the most reliable tests is the brine shrimp lethality test (BLST) that is simple procedure for screening of plant toxicity and bioactive compounds having antitumor activity. Material and methods: In the present study, aqueous extracts of Mentha logifolia, Achillea mellefolium, Mentha piperita, Matricaria recutita and Teucrium polium medicinal plants were screened for their cytotoxicity using brine shrimp lethality test. Five concentrations (25, 50, 75, 100, and 150 ppm) of each extract were applied in the bioassay and the LC50 determined. Results: The least LC50 value of 32 was obtained for hexane extract and the highest value of 13.87 was obtained for aqueous extract using the Finneys probit analysis which indicates high lethality of the extracts and presence of potent cytotoxic compounds in the plant extracts. Out of the 5 plants tested, M. recutita exhibited potent brine shrimp lethality with LC50 32 μg. M. logifolia, A. mellefolium, T. polium and M. piperita have also showed moderate cytotoxicity with LC50 111, 160, 172 and 247 μg respectively. Conclusion: The present study supports that brine shrimp bioassay is simple reliable and convenient method for evalution of bioactivity of medicinal plants and lends support for their use in traditional medicine.
Keywords: Artemia urmiana, Brine shrimp lethality test, Medicinal plants, Cytotoxicity doi:10.1016/j.clinbiochem.2011.08.896
Poster — [A-10-265-1] Beneficial effects of allicin on lead induced tissue enzyme alterations in common carp (Cyprinus carpio) Hasan Baghshani, Davar Shahsavani Ferdowsi University of Mashhad, Mashhad, Iran E-mail addresses: [email protected] (H. Baghshani), [email protected] (D. Shahsavani) Introduction: In toxicological studies, the alterations in the enzymatic activities directly reflect the metabolic disturbances and cell damage in specific organs. The aim of the current study was to determine the effects of lead exposure on tissue enzyme activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in selected tissues of common carp and also to evaluate the beneficial effects of allicin, the main active constituent of garlic, on modulating enzyme alterations. Methods: Common carp (Cyprinus carpio; total, n = 60) were divided randomly into three groups of 20 each. Group I fish were reared in normal freshwater and served as the control. Group II fish were exposed to lead acetate (7.0 mg L−1) for 10 days. Group III fish were exposed to lead acetate (7.0 mg L−1) for 10 days and also received dietary allicin (10 mg/kg body weight, daily) during lead acetate treatment. At the end of each exposure, enzyme activities in prepared tissue homogenates were determined using colorimetric methods. Results: Lead exposure caused a significant elevation of AST, ALT and LDH in the liver, as compared to control group (P < 0.05). The AST, ALT, ALP and LDH levels in kidney homogenates were considerably increased following lead exposure, although these increases were only significant for ALT and ALP values. Significant increases of brain ALT and gill ALP activities were also observed following lead exposure. However, alterations in the activities of other measured enzymes in both brain and gill of experimental groups were not significant. Conclusion: Allicin supplementation was effective in attenuating the increase in Pb-induced tissue enzyme activities in the most organs; therefore it might have some protective and therapeutic effects on lead poisoning. However, much more extensive biochemical and toxicological research are needed for better understanding of the molecular basis of lead induced tissue enzyme alterations and ameliorative properties of allicin. Keywords: Lead poisoning, Allicin, Tissue enzyme activity, Common carp doi:10.1016/j.clinbiochem.2011.08.897
Poster — [A-10-274-1] Purification and immunogenic characterization of anti-LPS rabbit antibodies produced against Salmonella Typhimurium extracted lipopolysaccharide Ebrahimi Zadeh Waleada, Seyedlatif Mousavi Gargarib, Masoumeh Rajabi Bazlb, Mohammad Mohammadib a Shahed University, Qom, Iran b Tehran-qum Express Way, Shahed University, Iran E-mail addresses: [email protected] (E.Z. Walead), [email protected] (S.M. Gargari),
Abstracts
[email protected] (M. Rajabi Bazl), [email protected] (M. Mohammadi) Introduction: Salmonellae are anaerobic bacteria of the family Enterobacteriaceae. Salmonella enterica serovar Typhimurium is among the most common Salmonella serovars causing infections in human, livestock and poultry. O antigen (Lipopolysaccharide) is the dominant antigen, giving the bacteria ability to survive in intracellular conditions. In this study the S. typhimurium was neutralized with IgG purified from immunized rabbit with LPS and then used to challenge BALB/c mice. Methods: The O antigen from S. typhimurium was extracted with phenol-chloroform petroleum ether and analyzed with Tricine-SDSPAGE and LPS-Silver stain methods. Rabbits were immunized by LPS. Serum was isolated and antibody titer was assayed by ELISA. Antibody is extracted by ammonium sulfate precipitation followed by purification with DEAE cellulose chromatography. Attachment ability of antibody to LPS and S. typhimurium was analyzed by ELISA. S. typhimurium was incubated with antibody for 1 h at 37 °C and then gavaged into BALB/c female mice stomach. Control mice were only given same amount of bacterium. Results: Extracted LPS showed similar pattern compared to standard LPS in Tricine-SDS-PAGE. Antibody showed great affinity to extracted LPS and S. typhimurium. Compared to control mice, when the bacteria were treated with antibody the CFU for LD50 was significantly increased. Conclusion: Using bacterium as an antigen in the ELISA test, we showed that the affinity of antibody to S. typhimurium bacterium did not reduced compared to LPS. Neutralization test showed that produced antibody is able to attach to the bacterium and inhibit its colonization in in vivo experiment. Keywords: Salmonella Typhimurium, LPS, Antibody, LD50, BALB/c mice doi:10.1016/j.clinbiochem.2011.08.898
Poster — [A-10-275-1] Heavy chain camelied-antibodies (VHH) passively protect BALB/c mice against BoNT serotype E in neutralization test Hamid Bakherada, Latif Mousavi Gargaria, Masoumeh Rajabi Bazlb, Mohammad Mohammadic, Hamed Zarea, Leila Safaeea a Biology Department, Basic Science Faculty, Shahed University, Tehran, Iran b Biochemical Department, Medicine Faculty, Shahid Beheshti University, Tehran, Iran c Biology Department, Basic Science Faculty, Shahid Chamran University, Ahvaz, Iran E-mail addresses: [email protected] (H. Bakherad), [email protected] (L.M. Gargari), [email protected] (M. Rajabi Bazl), [email protected] (M. Mohammadi), [email protected] (H. Zare), [email protected] (L. Safaee) Introduction: Food intoxications caused by botulinium neurotoxins (BoNTs) are the most lethal poisoning known in the world. Thus the preventive and treatment measure are necessary particularly in the developing countries. In this study we investigated in vivo neutralization of BoNTE with Heavy Chain Camelied-Antibodies (VHH). Methods: VHH panning was done after library construction of the VHH gene amplified by nested PCR. High affinity colonies were cultured to produce VHH antibody. The recombinant VHH was purified by affinity chromatography using Ni-Nta Column. The LD50 for BoNTE was estimated. In vivo neutralization assay was done by mixing different concentrations of toxin with antibody and incubation for 1 h at 37 °C. The mixture was brought to 200 μl with PBS and injected to
S357
mice intraperitoneasly. The survival of mice was monitored after 24 h. Results: LD50 of the purified BoNTE toxin in our experiment was estimated to be around 2 ng per mouse. Results from neutralization tests for the VHH antibody showed a 100% survival of mice injected with a lethal dose of as high as 8 LD50 of BoNTE toxin (16 ng per mouse). Conclusions: The effect of the valency of the VHH antibody fragments on affinity and neutralizing capacity was the major goal of this study. The data obtained from our experiments highlight the realistic potential of novel recombinant antibody fragments to be used as a threupathic agent in order to reduce the lethality caused by BoNTE toxin. Keywords: BoNTE, VHH antibody, Neutralization
doi:10.1016/j.clinbiochem.2011.08.899
Poster — [A-10-323-1] Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species Gholamreza Karimia, Javad Behravanb, Fateme Mosaffab, Leila Hosseinzadehc, Bahrami Gholamrezac a Mashhad University of Medical Sciences, Faculty of Pharmacy, Mashhad, Iran b Pharmacy School, Mashhad, Iran c Pharmacy School, Kermanshah, Iran E-mail addresses: [email protected] (G. Karimi), [email protected] (J. Behravan), [email protected] (F. Mosaffa), [email protected] (L. Hosseinzadeh) Introduction: Doxorubicin (DOX) is a widely used chemotherapy agent. The major adverse effect of DOX treatment in cancer patients is the onset of cardiomyopathy and heart failure. Reactive oxygen species (ROS) are proposed to be responsible for DOX cardiotoxicity. Curcumin a natural compound extracted from Curcuma longa L. is known for its antioxidant properties. Therefore, we evaluated the effects of curcumin on apoptosis induced by DOX in cardiac muscle cells. Materials and methods: Detection of apoptosis was performed by Annexin V-FITC apoptosis detection kit with flow cytometry. Quantitative specific RNA expression was performed in one step with CYBR-green invitrogen kit. Intracellular ROS levels were examined using DCF-DA. Results: Preteatment with curcumin significantly increased DOXinduced apoptosis of cardiac muscle cells through down regulation of Bcl2, up-regulation of caspase-8 and caspase-9. The Bax/Bcl-2 ratio increased significantly after 1 h pretreatment with curcumin. Also curcumin increased ROS generation by DOX. In response to excess DOX, NF-κB was activated. However, curcumin was able to inhibit NF-κB activation. Conclusion: Our results indicated that pretreatment with nontoxic concentrations of curcumin sensitized H9c2 cells to DOX-mediated apoptosis by generation of ROS. Keywords: Doxorubicin, Curcumin, Apoptosis
doi:10.1016/j.clinbiochem.2011.08.900
Poster — [A-10-417-1] In vitro quantitative genotoxic analysis of two dentin bonding agents on primary human dental pulp cells Mohammad Ehsan Zangeneh, Glenn Oyong Manila, Philippines E-mail addresses: [email protected] (M.E. Zangeneh), [email protected] (G. Oyong)