Objective The objective of this study was to investigate arsenic induced changes in blood 8-aminolevulinic acid dehydratase (ALAD) after in vitro and in vivo exposure to this element and its response to co-administrat...Objective The objective of this study was to investigate arsenic induced changes in blood 8-aminolevulinic acid dehydratase (ALAD) after in vitro and in vivo exposure to this element and its response to co-administration of meso 2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA (MiADMSA) either individually or in combination. Methods Rat whole blood was exposed to varying concentrations (0.1, 0.2 and 0.5 mmol/L) of arsenic (III) or arsenic (V), to assess their effects on blood ALAD activity. Varying concentrations of MiADMSA and DMSA (0.1,0.5 and 1.0 mmol/L) were also tried in combination to determine its ability to mask the effect of arsenic induced (0.5 mmol/L) inhibition of blood ALAD in vitro. In vitro and in vivo experiments were also conducted to determine the effects of DMSA and MiADMSA either individually or in combination with arsenic, on blood ALAD activity and blood arsenic concentration. Results In vitro experiments showed significant inhibition of the enzyme activity when 0.1-0.5 mmol/L of arsenic (III and V) was used. Treatment with MiADMSA increased ALAD activity when blood was incubated at the concentration of 0.1 mmol/L arsenic (III) and 0.1 mmol/L MiADMSA. No effect of 0.1 mmol/L MiADMSA on ALAD activity was noticed when the arsenic concentration was increased to 0.2 and 0.5 mmol/L. Similarly, MiADMSA at a lower concentration (0.1 mmol/L) was partially effective in the turnover of ALAD activity against 0.5 mmol/L arsenic (III), but at two higher concentrations (0.5 and 1.0 mmol/L) a complete restoration of ALAD activity was observed. DMSA at all the three concentrations (0.1,0.5 and 1.0 mmol/L) was effective in restoring ALAD activity to the normal value. Conclusions The results thus suggest that arsenic has a distinct effect on ALAD activity. Another important toxicological finding of the present study, based on in vivo experiments further suggests that combined administration of DMSA and MiADMSA could be more beneficial for reducing blood ALAD inhibition and blood arsenic concentration than the individual treatment.展开更多
L-Serine is considered a functional amino acid in the central nervous system, and induces sedation and hypnotic effects in some animal models of acute and chronic stress. Accordingly, while L-serine is a candidate ant...L-Serine is considered a functional amino acid in the central nervous system, and induces sedation and hypnotic effects in some animal models of acute and chronic stress. Accordingly, while L-serine is a candidate anti-stress factor, the central mechanism of L-serine is not clear. The present study clarifies the action of L-serine using acute chick brain slices. We investigated the changes in some extracellular fluid amino acid concentrations in response to L-serine perfusion. Taurine concentration decreased while L-alanine concentration increased following L-serine perfusion. To examine the involvement of the taurine transporter, the effect of L-serine on the taurine concentration in the presence and absence of Na+ was also investigated. Na+ had no effect on taurine concentration induced by L-serine perfusion. These results suggest that L-serine has an ability to promote L-alanine synthesis facilitating the catabolism of taurine. In conclusion, L-serine modifies the metabolism of taurine and L-alanine in the extracellular space in chick brain.展开更多
Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation,microbial cell factories for l-serine production using non-model microorganisms have not been reported.In this study,a ...Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation,microbial cell factories for l-serine production using non-model microorganisms have not been reported.In this study,a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l-serine production.By blocking the degradation pathway while introducing an exporter EceamA from E.coli,l-serine titer in recombinant Z.mobilis was increased from 15.30 mg/L to 62.67 mg/L.It was further increased to 260.33 mg/L after enhancing the l-serine biosynthesis pathway.Then,536.70 mg/L l-serine was achieved by removing feedback inhibition with a SerA mutant,and an elevated titer of 687.67 mg/L was further obtained through increasing serB copies while enhancing the precursors.Finally,855.66 mg/L l-serine can be accumulated with the supplementation of the glutamate precursor.This work thus not only constructed an l-serine producer to help understand the bottlenecks limiting l-serine production in Z.mobilis for further improvement,but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.展开更多
Dehydration of serine/threonine residues necessitates the activity of a dehydratase enzyme(domain)during the biosynthesis of RiPP.Recently,it was reported that dehydration in the thioviridamide pathway relies on a dis...Dehydration of serine/threonine residues necessitates the activity of a dehydratase enzyme(domain)during the biosynthesis of RiPP.Recently,it was reported that dehydration in the thioviridamide pathway relies on a distinct dehydratase complex that showcases the activities of a phosphotransferase TvaC for serine/threonine phosphorylation and a lyase TvaD for subsequent phosphate elimination.Herein,we report that dehydration reactions in the pathway of lantibiotic cacaoidin involves a similar dehydratase complex,CaoK/CaoY.Remarkably,this dehydratase complex exhibits flexible enzymatic activity and tolerates significant variations in its substrate peptide sequence.By binding with the leader peptide(LP)sequence of precursor peptide CaoA,the dehydration reactions proceed in a directional manner from the C-terminus of the core peptide(CP)to the N-terminus,and C-terminally truncated variants of CP are acceptable.We show that fusing CaoK to CaoY in a 1:1 molar ratio enables the resulting enzyme CaoYK to exert enhanced dehydration activity.CaoK binds with the LP to improve its own solubility and to ensure the phosphate transfer activity,while CaoY functions in a manner independently of LP.This work advances our understanding of the dehydration process during cacaoidin formation,and provides useful enzymes and methods for the studies of the rapidly emerging RiPPs.展开更多
Fishes are excellent markers of the extent of pollution from heavy metals in aquatic environments given that they are found at various levels of the food chain.This study aimed to investigate the bioaccumulation of he...Fishes are excellent markers of the extent of pollution from heavy metals in aquatic environments given that they are found at various levels of the food chain.This study aimed to investigate the bioaccumulation of heavy metals(Zn,Pb,Cd,As,and Hg)as well as the activity of delta-aminolevulinic acid dehydratase(δ-ALA-D)in the livers of cat fishes(Clarias gariepinus)collected from three rivers(Donga,Ibi and Gindin-Dorowa)in Taraba State,Nigeria.The concentrations of heavy metals in the liver tissues were determined using an atomic absorption spectrophotometer in accordance with the method of AOAC(2019),while theδ-ALA-D activity was assayed using the method of Sassa(1982).Results revealed that only Zn and As were present in the liver samples from the three rivers.Pb was found only in the liver from Gindin-Dorowa at the concentration of 0.0012mg/kg which is not significant(P<0.05)when compared with other locations,while Hg and Cd were absent in all the liver samples.The liver sample from Gindin-Dorowa had the highest concentration of Zn(4.2500 mg/kg),followed by Ibi(3.2067 mg/kg),and Donga having the least(2.5500 mg/kg),which were all substantially(P<0.05)different from one another.However,there was no significant(P<0.05)difference in the As composition of liver from Donga(0.0013 mg/kg),Ibi(0.0012 mg/kg)and Gindin-Dorowa(0.0010 mg/kg).The result ofδ-ALA-D activity showed that the highest enzymatic activity was found in the liver sample from Donga which has the least Zn and no Pb content,followed by Ibi sample.This validates the report that heavy metals impairδ-ALA-D activity.Nonetheless,the concentrations of all metals in fish livers from all regions do not exceed the acceptable limits set by international law,making them safe for human consumption and possibly not having a negative impact on public health.Since there is little or no industrial activity in the studied locations,these levels may be consequent to low anthropogenic inputs.The current situation should be safeguarded to prevent pollution of the river’s aquatic biota in the near future,and more appropriate steps should be made to guarantee higher fish quality and life in the rivers.展开更多
Organoseleniums are a class of compounds attracting attention across the globe owing to their Glutathione peroxidase(GPx)mimicry,which confers on them a strong antioxidant activity.Diphenyl diselenide(DPDS)is an Organ...Organoseleniums are a class of compounds attracting attention across the globe owing to their Glutathione peroxidase(GPx)mimicry,which confers on them a strong antioxidant activity.Diphenyl diselenide(DPDS)is an Organoselenium whose GPx mimetic property has been suggested to rely on the oxidation of non-protein or protein thiols critical to the activities of some sulfhydryl enzymes.This study,therefore investigated the GPx mimic/antioxidant property of DPDS as well as the role of thiols of two key sulfhydryl enzymes,cerebral Na^(+)/K^(+)-ATPase(sodium pump)and hepatic delta-aminolevulinic acid dehydratase(δ-ALAD)in the GPx mimicry of DPDS.Albino Wistar rats were euthanized,and the liver and brain were removed and used to assay for the effect of DPDS on lipid peroxidation induced by two prooxidants[Fe2^(+)(10μM)and H2O2,(1 mM)]as well as the activities of the sulfhydryl enzymes.The results revealed that DPDS profoundly(P<0.05)counteracted Fe2^(+)and H2O2-induced lipid peroxidation in the rats’hepatic and cerebral tissues.Furthermore,the results of assay systems for lipid peroxidation and sodium pump revealed that DPDS inhibited Na^(+)/K^(+)-ATPase and lipid peroxidation in the brain tissue homogenates in the same reaction system.A similar result was obtained in the assay system for lipid peroxidation and hepaticδ-ALAD as DPDS simultaneously inhibited the enzyme’s activity and lipid peroxidation.This suggests that the GPx mimetic property of DPDS may be linked to the enzymes’loss of activity,which further validates the suggestions that the enzymes’inhibition,as well as the antioxidant action of DPDS,rely on the oxidation of critical thiols of the enzymes.However,the GPx mimicry of DPDS should be investigated in the presence of thiol-blocking or oxidizing agents in biological systems in order to further ascertain the role of protein thiols.展开更多
基因 cai B和基因 cai E分别编码肉碱脱水酶及其辅因子合成酶 ,携带这两个基因的重组菌可以共表达两个外源蛋白 ,得到高活性肉碱脱水酶。分别构建这两个基因的表达质粒 p ET2 8cai B和 p ET2 2 cai E,利用双抗生素筛选法 ,获得能稳定遗...基因 cai B和基因 cai E分别编码肉碱脱水酶及其辅因子合成酶 ,携带这两个基因的重组菌可以共表达两个外源蛋白 ,得到高活性肉碱脱水酶。分别构建这两个基因的表达质粒 p ET2 8cai B和 p ET2 2 cai E,利用双抗生素筛选法 ,获得能稳定遗传的双质粒转化子。经 IPTG诱导 ,两个基因共表达 ,表达量分别占菌体总蛋白的 39%和 2 0 % ,共转化菌的酶活力比 p ET2 8cai B单转化菌提高了 2 .3倍 。展开更多
文摘Objective The objective of this study was to investigate arsenic induced changes in blood 8-aminolevulinic acid dehydratase (ALAD) after in vitro and in vivo exposure to this element and its response to co-administration of meso 2,3-dimercaptosuccinic acid (DMSA) and monoisoamyl DMSA (MiADMSA) either individually or in combination. Methods Rat whole blood was exposed to varying concentrations (0.1, 0.2 and 0.5 mmol/L) of arsenic (III) or arsenic (V), to assess their effects on blood ALAD activity. Varying concentrations of MiADMSA and DMSA (0.1,0.5 and 1.0 mmol/L) were also tried in combination to determine its ability to mask the effect of arsenic induced (0.5 mmol/L) inhibition of blood ALAD in vitro. In vitro and in vivo experiments were also conducted to determine the effects of DMSA and MiADMSA either individually or in combination with arsenic, on blood ALAD activity and blood arsenic concentration. Results In vitro experiments showed significant inhibition of the enzyme activity when 0.1-0.5 mmol/L of arsenic (III and V) was used. Treatment with MiADMSA increased ALAD activity when blood was incubated at the concentration of 0.1 mmol/L arsenic (III) and 0.1 mmol/L MiADMSA. No effect of 0.1 mmol/L MiADMSA on ALAD activity was noticed when the arsenic concentration was increased to 0.2 and 0.5 mmol/L. Similarly, MiADMSA at a lower concentration (0.1 mmol/L) was partially effective in the turnover of ALAD activity against 0.5 mmol/L arsenic (III), but at two higher concentrations (0.5 and 1.0 mmol/L) a complete restoration of ALAD activity was observed. DMSA at all the three concentrations (0.1,0.5 and 1.0 mmol/L) was effective in restoring ALAD activity to the normal value. Conclusions The results thus suggest that arsenic has a distinct effect on ALAD activity. Another important toxicological finding of the present study, based on in vivo experiments further suggests that combined administration of DMSA and MiADMSA could be more beneficial for reducing blood ALAD inhibition and blood arsenic concentration than the individual treatment.
文摘L-Serine is considered a functional amino acid in the central nervous system, and induces sedation and hypnotic effects in some animal models of acute and chronic stress. Accordingly, while L-serine is a candidate anti-stress factor, the central mechanism of L-serine is not clear. The present study clarifies the action of L-serine using acute chick brain slices. We investigated the changes in some extracellular fluid amino acid concentrations in response to L-serine perfusion. Taurine concentration decreased while L-alanine concentration increased following L-serine perfusion. To examine the involvement of the taurine transporter, the effect of L-serine on the taurine concentration in the presence and absence of Na+ was also investigated. Na+ had no effect on taurine concentration induced by L-serine perfusion. These results suggest that L-serine has an ability to promote L-alanine synthesis facilitating the catabolism of taurine. In conclusion, L-serine modifies the metabolism of taurine and L-alanine in the extracellular space in chick brain.
基金This work was supported by the National Key Research and Development Program of China(2022YFA0911800)National Natural Science Foundation of China(CN)(21978071)+2 种基金the Key Science and Technology Innovation Project of Hubei Province(2021BAD001)the Innovation Base for Introducing Talents of Discipline of Hubei Province(2019BJH021)We also acknowledge the support from the State Key Laboratory of Biocatalysis and Enzyme Engineering。
文摘Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation,microbial cell factories for l-serine production using non-model microorganisms have not been reported.In this study,a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l-serine production.By blocking the degradation pathway while introducing an exporter EceamA from E.coli,l-serine titer in recombinant Z.mobilis was increased from 15.30 mg/L to 62.67 mg/L.It was further increased to 260.33 mg/L after enhancing the l-serine biosynthesis pathway.Then,536.70 mg/L l-serine was achieved by removing feedback inhibition with a SerA mutant,and an elevated titer of 687.67 mg/L was further obtained through increasing serB copies while enhancing the precursors.Finally,855.66 mg/L l-serine can be accumulated with the supplementation of the glutamate precursor.This work thus not only constructed an l-serine producer to help understand the bottlenecks limiting l-serine production in Z.mobilis for further improvement,but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.
基金supported in part by grants from the National Key Research and Development Program of China(2022YFC2303100 for L.P and W.L)the National Natural Science Foundation of China(32030002 and 22193070 for W.L)+1 种基金the Science and Technology Commission of Shanghai Municipality(20XD1425200 for L.P)the CAS Youth Interdisciplinary Team(JCTD-2022-10 for L.P).
文摘Dehydration of serine/threonine residues necessitates the activity of a dehydratase enzyme(domain)during the biosynthesis of RiPP.Recently,it was reported that dehydration in the thioviridamide pathway relies on a distinct dehydratase complex that showcases the activities of a phosphotransferase TvaC for serine/threonine phosphorylation and a lyase TvaD for subsequent phosphate elimination.Herein,we report that dehydration reactions in the pathway of lantibiotic cacaoidin involves a similar dehydratase complex,CaoK/CaoY.Remarkably,this dehydratase complex exhibits flexible enzymatic activity and tolerates significant variations in its substrate peptide sequence.By binding with the leader peptide(LP)sequence of precursor peptide CaoA,the dehydration reactions proceed in a directional manner from the C-terminus of the core peptide(CP)to the N-terminus,and C-terminally truncated variants of CP are acceptable.We show that fusing CaoK to CaoY in a 1:1 molar ratio enables the resulting enzyme CaoYK to exert enhanced dehydration activity.CaoK binds with the LP to improve its own solubility and to ensure the phosphate transfer activity,while CaoY functions in a manner independently of LP.This work advances our understanding of the dehydration process during cacaoidin formation,and provides useful enzymes and methods for the studies of the rapidly emerging RiPPs.
文摘Fishes are excellent markers of the extent of pollution from heavy metals in aquatic environments given that they are found at various levels of the food chain.This study aimed to investigate the bioaccumulation of heavy metals(Zn,Pb,Cd,As,and Hg)as well as the activity of delta-aminolevulinic acid dehydratase(δ-ALA-D)in the livers of cat fishes(Clarias gariepinus)collected from three rivers(Donga,Ibi and Gindin-Dorowa)in Taraba State,Nigeria.The concentrations of heavy metals in the liver tissues were determined using an atomic absorption spectrophotometer in accordance with the method of AOAC(2019),while theδ-ALA-D activity was assayed using the method of Sassa(1982).Results revealed that only Zn and As were present in the liver samples from the three rivers.Pb was found only in the liver from Gindin-Dorowa at the concentration of 0.0012mg/kg which is not significant(P<0.05)when compared with other locations,while Hg and Cd were absent in all the liver samples.The liver sample from Gindin-Dorowa had the highest concentration of Zn(4.2500 mg/kg),followed by Ibi(3.2067 mg/kg),and Donga having the least(2.5500 mg/kg),which were all substantially(P<0.05)different from one another.However,there was no significant(P<0.05)difference in the As composition of liver from Donga(0.0013 mg/kg),Ibi(0.0012 mg/kg)and Gindin-Dorowa(0.0010 mg/kg).The result ofδ-ALA-D activity showed that the highest enzymatic activity was found in the liver sample from Donga which has the least Zn and no Pb content,followed by Ibi sample.This validates the report that heavy metals impairδ-ALA-D activity.Nonetheless,the concentrations of all metals in fish livers from all regions do not exceed the acceptable limits set by international law,making them safe for human consumption and possibly not having a negative impact on public health.Since there is little or no industrial activity in the studied locations,these levels may be consequent to low anthropogenic inputs.The current situation should be safeguarded to prevent pollution of the river’s aquatic biota in the near future,and more appropriate steps should be made to guarantee higher fish quality and life in the rivers.
文摘Organoseleniums are a class of compounds attracting attention across the globe owing to their Glutathione peroxidase(GPx)mimicry,which confers on them a strong antioxidant activity.Diphenyl diselenide(DPDS)is an Organoselenium whose GPx mimetic property has been suggested to rely on the oxidation of non-protein or protein thiols critical to the activities of some sulfhydryl enzymes.This study,therefore investigated the GPx mimic/antioxidant property of DPDS as well as the role of thiols of two key sulfhydryl enzymes,cerebral Na^(+)/K^(+)-ATPase(sodium pump)and hepatic delta-aminolevulinic acid dehydratase(δ-ALAD)in the GPx mimicry of DPDS.Albino Wistar rats were euthanized,and the liver and brain were removed and used to assay for the effect of DPDS on lipid peroxidation induced by two prooxidants[Fe2^(+)(10μM)and H2O2,(1 mM)]as well as the activities of the sulfhydryl enzymes.The results revealed that DPDS profoundly(P<0.05)counteracted Fe2^(+)and H2O2-induced lipid peroxidation in the rats’hepatic and cerebral tissues.Furthermore,the results of assay systems for lipid peroxidation and sodium pump revealed that DPDS inhibited Na^(+)/K^(+)-ATPase and lipid peroxidation in the brain tissue homogenates in the same reaction system.A similar result was obtained in the assay system for lipid peroxidation and hepaticδ-ALAD as DPDS simultaneously inhibited the enzyme’s activity and lipid peroxidation.This suggests that the GPx mimetic property of DPDS may be linked to the enzymes’loss of activity,which further validates the suggestions that the enzymes’inhibition,as well as the antioxidant action of DPDS,rely on the oxidation of critical thiols of the enzymes.However,the GPx mimicry of DPDS should be investigated in the presence of thiol-blocking or oxidizing agents in biological systems in order to further ascertain the role of protein thiols.
