A strain capable of phenol degradation, hetemtrophic nitrification and aerobic denitrification was isolated from activated sludge of coking-plant wastewater ponds under aerobic condition. Based on its morphology, phys...A strain capable of phenol degradation, hetemtrophic nitrification and aerobic denitrification was isolated from activated sludge of coking-plant wastewater ponds under aerobic condition. Based on its morphology, physiology, biochemical analysis and phylogenetic characteristics, the isolate was identified as Diaphorobacter sp. PD-7. Biodegradation tests of phenol showed that the maximum phenol degradation occurred at the late phase of exponential growth stages, with 1400 mg·L^-1 phenol completely degraded within 85 h. Diaphorobacter sp. PD-7 accumulated a vast quantity of phenol hydroxylase in this physiological phase, ensuring that the cells quickly utilize phenol as a sole carbon and energy source. The kinetic behavior ofDiaphorobacter sp. PD-7 in batch cultures was investigated over a wide range of initial phenol concentrations （0-1400mg·L^-1） by using the Haldane model, which adequately describes the dynamic behavior of phenol biodegradation by strain Diaphombacter sp. PD-7. At initial phenol concentration of 1400mg· L^-l, batch experiments （0.25 L flask） of nitrogen removal under aerobic condition gave almost entirely removal of 120.69mg· L^- 1 ammonium nitrogen within 75 h, while nitrate nitrogen removal reached 91% within 65 h. Moreover, hydroxylamine oxidase, periplasmic nitrate reductase and nitrite reductase were successfully expressed in the isolate.展开更多
Carboxylesterases （CarEs） from two field populations of the oriental migratory locust, Locusta migratoria manilensis （Meyen）, were examined to try to understand their contribution to malathion insensitivity. The C...Carboxylesterases （CarEs） from two field populations of the oriental migratory locust, Locusta migratoria manilensis （Meyen）, were examined to try to understand their contribution to malathion insensitivity. The CarEs activities in Wudi population （WD） were 1.75- and 1.50-fold significantly higher than those in Huangliu population （HL） when a-naphthyl acetate （a-NA） and [3-naphthyl acetate were used as substrates, respectively. Such elevated CarEs activities presented in the WD could be because of an increased staining intensity of the a-NA-hydrolyzing CarEs as shown on the nondenaturing polyacrylamide gel electrophoresis. Inhibition studies of CarEs using paraoxon and malaoxon indicated that CarE activities in the HL were more strongly inhibited than those in the WD. Furthermore, a 449-bp DNA fragment of CarE was obtained from L. migratoria manilensis. Hemiquantity reverse transcription-polymerase chain reaction analysis showed that CarE gene expression level in the WD was higher than that in the HL. The higher CarE activities and the increased CarE mRNA level in the WD appeared to be associated with decreased susceptibility to malathion in the WD due to the application of organophosphorus insecticides.展开更多
Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molec...Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate （4MU-（GlcNAe）3）, and higher Km value for the longer substrate （CM-Chitin- RBV） compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain （CBD） tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5- I and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-（GlcNAc）3, whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBE These findings are helpful for further research to clarit}g their different roles in insect growth and development.展开更多
In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (XinxiangCity, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when co...In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (XinxiangCity, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when concentrations of ATCincreased, showing a characteristic phenomenon of substrate inhibition at high concentration in both populations. Suchinhibition occurred at relatively low concentration for AChE from Xinxiang population but relatively high for AChE fromChangzhi population. The kinetic study showed that there were no significant differences between the two populations inthe Km values. The Km value in Changzhi population was only 1.09-fold higher than that in Xinxiang population. However,significant differences were observed between the two populations in Vmax values. The Vmax value in Changzhi populationwas 1.32-fold higher than that in Xinxiang population. The inhibition study in vitro showed that the AChE from bothpopulations exhibited similar rank order in sensitivity to inhibition by three OPs, as determined by comparison of theirbimolecular rate constants (ki), from the most potent inhibition to the least was chlopyrifos-oxon > paraoxon >demeton-s-methyl for AChE from the two populations and that the ki values in Xinxiang population were lower than those in Changzhipopulation. The I50 values of AChE from Xinxiang population were 4.84-, 2.66-, and 1.92-fold less sensitive to inhibition byparaoxon, chlopyrifos-oxon, and demeton-s-methyl. These results were consistent with the results in bioassay. It isinferred that AChE insensitivity to OP insecticides plays an important role in the differences of insusceptibility of Oxyachinensis to malathion between the two populations.展开更多
The susceptibility of Oxya chinensis to malathion was compared in larvae and adults from a field population, collected from Jinyuan outskirt, Shanxi Province. The results showed that Oxya chinensis was more suscepti...The susceptibility of Oxya chinensis to malathion was compared in larvae and adults from a field population, collected from Jinyuan outskirt, Shanxi Province. The results showed that Oxya chinensis was more susceptible to malathion in the adult stage than in the larval stage. The LD50 values for malathion susceptibility of Oxya chinensis were 4.94 and 2.44 mg g-1 body weight in the larvae and adults respectively. The results indicated that the larvae were 2.02-fold less susceptible to malathion than the adults. The general esterases and the kinetics were characterized and compared between the two life stages and between females and males. Larval preparations of Oxya chinensis were more active than adult preparations in females and males. The larvae showed 1.18-, 1.49-, and 1.17- fold higher specific activities than the adults in females with α-NA, α-NB and β-NA respectively. In males, the ratios were 1.34-, 1.70-, and 1.06-fold. Female preparations were more active than those of males in the adults. The reverse results were observed in the larvae where male preparations were more active than female preparations. Kinetic studies showed that Km values of general esterases hydrolyzing α-NA, α-NB, and β-NA in the adult stage were 1.36-, 1.32- and 1.39-fold respectively, higher than those in the larval stage in females. In males, the ratios were 1.24-, 2.14-, and 1.20-fold. The esterase from male insects had a higher affinity (lower Km value) to the substrate than those from females. The results also showed that the Vmax values of general esterase hydrolyzing α-NA, α-NB, and β-NA in the two stages were similar. From the results of bioassays and biochemical analyses, it has been inferred that a higher level of resistance to malathion in larvae than in adults would appear to result from differences in the expression of resistance mechanisms in these two life stages. Enhanced esterase activities appeared to play a major role in resistance to malathion in both larvae and adults. From the an展开更多
本文从人肝、骨、小肠和胎盘组织中分别提取制备肝、骨、小肠和胎盘型碱性磷酸酶。并对这4种同工酶的动力学进行了系统研究。根据胎盘型碱性磷酸酶在56℃×2 h 活性不变的热稳定性,及其 Km 为4.09×10^(-5)mol/L、激活剂是1.2 m...本文从人肝、骨、小肠和胎盘组织中分别提取制备肝、骨、小肠和胎盘型碱性磷酸酶。并对这4种同工酶的动力学进行了系统研究。根据胎盘型碱性磷酸酶在56℃×2 h 活性不变的热稳定性,及其 Km 为4.09×10^(-5)mol/L、激活剂是1.2 mmol/LMg^(2+)和最适 pH10.5等的动力学参数,设计建立了一种以 Mg^(2+)作为激活剂的胎盘性碱性磷酸酶荧光测定法。本法的灵敏度比 Miggiano 的 EDTA抑制剂法提高约60倍,准确性达99.8±1.4%,特异性达98.3±1.5%,组内变异系数为1.7%;适于临床推广应用。展开更多
AIM： To investigate the immunogenicity of H pylori proteins, to evaluate the production rate of anti H pylori IgG antibodies in relation to time and to demonstrate the fidelity of newly optimized in-house enzymelinke...AIM： To investigate the immunogenicity of H pylori proteins, to evaluate the production rate of anti H pylori IgG antibodies in relation to time and to demonstrate the fidelity of newly optimized in-house enzymelinked immunosorbent assay （ELISA） technique as an alternative for H pylori infection assay. METHODS： In the present study, 100 μg of formalinfixed H pylori whole cell antigens was injected into an experimental animal （New Zealand white female rabbit） intramuscularly on d 0, 16, 27 and 35. The first two doses were injected with adjuvants. On d 0, a serum sample was collected from the rabbit before immunization and this pre-immunized serum was used as a negative control for the whole study. To evaluate the immunogenic responses of the injected antigen, serum samples were collected from the rabbit at regular intervals up to d 42. The sera were analyzed using inhouse ELISA and Western blot techniques.RESULTS： The production of anti Hpylor/IgG antibodies in the rabbit in response to the injected antigen increased almost exponentially up to d 14 and after that it was maintained at the same level until the last day （d 42）. By analyzing the immune profiles of immunized sera, 11 proteins were identified to be immunogenic, among them 2 （approximately 100 kDa and 85 kDa） were most prominent. CONCLUSION： Analysis of the immune responses against pathogenic microorganisms like H py/ori is necessary for the development of various diagnostic and preventive approaches. The results of this experiment reveal that the formalin-fixed H pylori whole cell antigens injected into the rabbit are highly immunogenic. These prominent proteins （approximately 100 kDa and 85 kDa） might have higher immunogenic effects among humans infected with H pylori and some of these immunogenic proteins can be included in diagnostic approaches based on serology and also for vaccine formulation. The in- house ELISA is a promising alternative compared to invasive techniques.展开更多
基金the National Natural Science Foundation of China(51378330 and51408396)the Natural Science Foundation of Shanxi Province(2013021023-3)
文摘A strain capable of phenol degradation, hetemtrophic nitrification and aerobic denitrification was isolated from activated sludge of coking-plant wastewater ponds under aerobic condition. Based on its morphology, physiology, biochemical analysis and phylogenetic characteristics, the isolate was identified as Diaphorobacter sp. PD-7. Biodegradation tests of phenol showed that the maximum phenol degradation occurred at the late phase of exponential growth stages, with 1400 mg·L^-1 phenol completely degraded within 85 h. Diaphorobacter sp. PD-7 accumulated a vast quantity of phenol hydroxylase in this physiological phase, ensuring that the cells quickly utilize phenol as a sole carbon and energy source. The kinetic behavior ofDiaphorobacter sp. PD-7 in batch cultures was investigated over a wide range of initial phenol concentrations （0-1400mg·L^-1） by using the Haldane model, which adequately describes the dynamic behavior of phenol biodegradation by strain Diaphombacter sp. PD-7. At initial phenol concentration of 1400mg· L^-l, batch experiments （0.25 L flask） of nitrogen removal under aerobic condition gave almost entirely removal of 120.69mg· L^- 1 ammonium nitrogen within 75 h, while nitrate nitrogen removal reached 91% within 65 h. Moreover, hydroxylamine oxidase, periplasmic nitrate reductase and nitrite reductase were successfully expressed in the isolate.
基金the National Natural Science Foundation of China （30470219）.
文摘Carboxylesterases （CarEs） from two field populations of the oriental migratory locust, Locusta migratoria manilensis （Meyen）, were examined to try to understand their contribution to malathion insensitivity. The CarEs activities in Wudi population （WD） were 1.75- and 1.50-fold significantly higher than those in Huangliu population （HL） when a-naphthyl acetate （a-NA） and [3-naphthyl acetate were used as substrates, respectively. Such elevated CarEs activities presented in the WD could be because of an increased staining intensity of the a-NA-hydrolyzing CarEs as shown on the nondenaturing polyacrylamide gel electrophoresis. Inhibition studies of CarEs using paraoxon and malaoxon indicated that CarE activities in the HL were more strongly inhibited than those in the WD. Furthermore, a 449-bp DNA fragment of CarE was obtained from L. migratoria manilensis. Hemiquantity reverse transcription-polymerase chain reaction analysis showed that CarE gene expression level in the WD was higher than that in the HL. The higher CarE activities and the increased CarE mRNA level in the WD appeared to be associated with decreased susceptibility to malathion in the WD due to the application of organophosphorus insecticides.
基金Acknowledgments This work was supported by the National Basic Research Program of China （2012CB114102）, National Natural Science Foundation of China （31272380）.
文摘Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate （4MU-（GlcNAe）3）, and higher Km value for the longer substrate （CM-Chitin- RBV） compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain （CBD） tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5- I and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-（GlcNAc）3, whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBE These findings are helpful for further research to clarit}g their different roles in insect growth and development.
基金supported by National Natural Science Foundation of China(30170612)Science and Technology Commission of Shanxi Province(041005)to Ma Enbo
文摘In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (XinxiangCity, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when concentrations of ATCincreased, showing a characteristic phenomenon of substrate inhibition at high concentration in both populations. Suchinhibition occurred at relatively low concentration for AChE from Xinxiang population but relatively high for AChE fromChangzhi population. The kinetic study showed that there were no significant differences between the two populations inthe Km values. The Km value in Changzhi population was only 1.09-fold higher than that in Xinxiang population. However,significant differences were observed between the two populations in Vmax values. The Vmax value in Changzhi populationwas 1.32-fold higher than that in Xinxiang population. The inhibition study in vitro showed that the AChE from bothpopulations exhibited similar rank order in sensitivity to inhibition by three OPs, as determined by comparison of theirbimolecular rate constants (ki), from the most potent inhibition to the least was chlopyrifos-oxon > paraoxon >demeton-s-methyl for AChE from the two populations and that the ki values in Xinxiang population were lower than those in Changzhipopulation. The I50 values of AChE from Xinxiang population were 4.84-, 2.66-, and 1.92-fold less sensitive to inhibition byparaoxon, chlopyrifos-oxon, and demeton-s-methyl. These results were consistent with the results in bioassay. It isinferred that AChE insensitivity to OP insecticides plays an important role in the differences of insusceptibility of Oxyachinensis to malathion between the two populations.
基金supported by the National Natura1 Science Foundation of China(30170612)the Science and Technology Commission of Shanxi Province(041005)to MA Enbo.
文摘The susceptibility of Oxya chinensis to malathion was compared in larvae and adults from a field population, collected from Jinyuan outskirt, Shanxi Province. The results showed that Oxya chinensis was more susceptible to malathion in the adult stage than in the larval stage. The LD50 values for malathion susceptibility of Oxya chinensis were 4.94 and 2.44 mg g-1 body weight in the larvae and adults respectively. The results indicated that the larvae were 2.02-fold less susceptible to malathion than the adults. The general esterases and the kinetics were characterized and compared between the two life stages and between females and males. Larval preparations of Oxya chinensis were more active than adult preparations in females and males. The larvae showed 1.18-, 1.49-, and 1.17- fold higher specific activities than the adults in females with α-NA, α-NB and β-NA respectively. In males, the ratios were 1.34-, 1.70-, and 1.06-fold. Female preparations were more active than those of males in the adults. The reverse results were observed in the larvae where male preparations were more active than female preparations. Kinetic studies showed that Km values of general esterases hydrolyzing α-NA, α-NB, and β-NA in the adult stage were 1.36-, 1.32- and 1.39-fold respectively, higher than those in the larval stage in females. In males, the ratios were 1.24-, 2.14-, and 1.20-fold. The esterase from male insects had a higher affinity (lower Km value) to the substrate than those from females. The results also showed that the Vmax values of general esterase hydrolyzing α-NA, α-NB, and β-NA in the two stages were similar. From the results of bioassays and biochemical analyses, it has been inferred that a higher level of resistance to malathion in larvae than in adults would appear to result from differences in the expression of resistance mechanisms in these two life stages. Enhanced esterase activities appeared to play a major role in resistance to malathion in both larvae and adults. From the an
基金Research Funds of Microbiology Department, Dhaka University
文摘AIM： To investigate the immunogenicity of H pylori proteins, to evaluate the production rate of anti H pylori IgG antibodies in relation to time and to demonstrate the fidelity of newly optimized in-house enzymelinked immunosorbent assay （ELISA） technique as an alternative for H pylori infection assay. METHODS： In the present study, 100 μg of formalinfixed H pylori whole cell antigens was injected into an experimental animal （New Zealand white female rabbit） intramuscularly on d 0, 16, 27 and 35. The first two doses were injected with adjuvants. On d 0, a serum sample was collected from the rabbit before immunization and this pre-immunized serum was used as a negative control for the whole study. To evaluate the immunogenic responses of the injected antigen, serum samples were collected from the rabbit at regular intervals up to d 42. The sera were analyzed using inhouse ELISA and Western blot techniques.RESULTS： The production of anti Hpylor/IgG antibodies in the rabbit in response to the injected antigen increased almost exponentially up to d 14 and after that it was maintained at the same level until the last day （d 42）. By analyzing the immune profiles of immunized sera, 11 proteins were identified to be immunogenic, among them 2 （approximately 100 kDa and 85 kDa） were most prominent. CONCLUSION： Analysis of the immune responses against pathogenic microorganisms like H py/ori is necessary for the development of various diagnostic and preventive approaches. The results of this experiment reveal that the formalin-fixed H pylori whole cell antigens injected into the rabbit are highly immunogenic. These prominent proteins （approximately 100 kDa and 85 kDa） might have higher immunogenic effects among humans infected with H pylori and some of these immunogenic proteins can be included in diagnostic approaches based on serology and also for vaccine formulation. The in- house ELISA is a promising alternative compared to invasive techniques.
