Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions includ...Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.展开更多
Background There is a need for biomarkers to support an accurate diagnosis of Parkinson’s disease(PD).Cerebrospinal fluid(CSF)has been a successful biofluid for finding neurodegenerative biomarkers,and modern highly ...Background There is a need for biomarkers to support an accurate diagnosis of Parkinson’s disease(PD).Cerebrospinal fluid(CSF)has been a successful biofluid for finding neurodegenerative biomarkers,and modern highly sensitive multiplexing methods offer the possibility to perform discovery studies.Using a large-scale multiplex proximity extension assay(PEA)approach,we aimed to discover novel diagnostic protein biomarkers allowing accurate discrimination of PD from both controls and atypical Parkinsonian disorders(APD).Methods CSF from patients with PD,corticobasal syndrome(CBS),progressive supranuclear palsy(PSP),multiple system atrophy and controls,were analysed with Olink PEA panels.Three cohorts were used in this study,comprising 192,88 and 36 cases,respectively.All samples were run on the Cardiovascular II,Oncology II and Metabolism PEA panels.Results Our analysis revealed that 26 and 39 proteins were differentially expressed in the CSF of test and validation PD cohorts,respectively,compared to controls.Among them,6 proteins were changed in both cohorts.Midkine(MK)was increased in PD with the strongest effect size and results were validated with ELISA.Another most increased protein in PD,DOPA decarboxylase(DDC),which catalyses the decarboxylation of DOPA(L-3,4-dihydroxyphenylalanine)to dopamine,was strongly correlated with dopaminergic treatment.Moreover,Kallikrein 10 was specifically changed in APD compared with both PD and controls,but unchanged between PD and controls.Wnt inhibitory factor 1 was consistently downregulated in CBS and PSP patients in two independent cohorts.Conclusions Using the large-scale PEA approach,we have identified potential novel PD diagnostic biomarkers,most notably MK and DDC,in the CSF of PD patients.展开更多
目的:通过对胃腺癌患者的腹腔灌洗液检测多巴脱羧酶(dopa decarboxylase D D C)m R N A表达,评估D D C成为预测胃腺癌腹膜微转移的新指标的可能性.方法:收集南昌大学第二附属医院胃肠外科87例胃腺癌患者的腹腔灌洗液,应用实时荧光定量...目的:通过对胃腺癌患者的腹腔灌洗液检测多巴脱羧酶(dopa decarboxylase D D C)m R N A表达,评估D D C成为预测胃腺癌腹膜微转移的新指标的可能性.方法:收集南昌大学第二附属医院胃肠外科87例胃腺癌患者的腹腔灌洗液,应用实时荧光定量聚合酶链反应(quantitative real time polymerase chain reaction,QRT-PCR方法相对定量检测并比较DDC m RNA的表达.12例非癌症患者腹腔灌洗液作为阴性对照.结果:87例胃腺癌患者中,T1有6例,T2有14例,T3有28例,T4有39例,DDC m RNA的表达在不同T分级(浸润深度分级)DDC m RNA的相对表达值(×107)分别为:T1,168±21T2,283±87;T3,31162±4261;T4,35310±6593.非癌症组腹腔灌洗液中,DDC m RNA相对表达值(×107)为:60.28±19.00.此外D D C的表达还与组织分化程度、病理分化类型、是否有淋巴结转移等有关系.胃腺癌腹腔灌洗液应用常规腹腔细胞学检查(conventional intraperitoneal cytology,CY)检查,11例阳性(CY+),阳性率为13%(11/87).11例(CY+)中有9例DDC m RNA表达相对值结果高于临界值,归类于DDC+,DDC的敏感性为86%(9/11),此外,在10例T1患者和14例T2患者中DDC+为2例,且非癌症组中均未见DDC+,DDC的特异性为92%(22/24).表明腹腔灌洗液中DDC在胃腺癌不同浸润深度下差异性表达(P<0.05),具有较好的敏感性和特异性.结论:应用QRT-PCR技术可以有效检测腹腔灌洗液中DDC m RNA表达,DDC可能成为预测胃癌腹膜转移的可靠指标.展开更多
Monoamine neurotransmitters play an important role in the modulation of sensory, motor and autonomic functions in the spinal cord. Although traditionally it is believed that in mammalian spinal cord, monoamine neurotr...Monoamine neurotransmitters play an important role in the modulation of sensory, motor and autonomic functions in the spinal cord. Although traditionally it is believed that in mammalian spinal cord, monoamine neurotransmitters mainly originate from the brain, accumulating evidence indicates that especially when the spinal cord is injured, they can also be produced in the spinal cord. In this review, I will present evidence for a possible pathway for two-step synthesis of dopamine and serotonin in the spinal cord. Published data from different sources and unpublished data from my own ongoing projects indicate that monoenzymatic cells expressing aromatic L-amino acid decarboxylase（AADC）, tyrosine hydroxylase（TH） or tryptophan hydroxylase（TPH） are present in the spinal cord and that these TH and THP cells often lie in close proximity to AADC cells. Prompted by the above evidence, I hypothesize that dopamine and serotonin could be synthesized sequentially in two monoenzymatic cells in the spinal cord via a TH-AADC and a TPH-AADC cascade respectively. The monoamines synthesized through this pathway may compensate for lost neurotransmitters following spinal cord injury and also may play specific roles in the recovery of sensory, motor and autonomic functions.展开更多
The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ont...The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes （TH and DDC）, in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.展开更多
Melanin is involved in cuticle pigmentation and sclerotization of insects,which is critical for maintaining structural integrity and functional completeness of insect cuti-cle.The 2 key enzymes of tyrosine hydroxylase...Melanin is involved in cuticle pigmentation and sclerotization of insects,which is critical for maintaining structural integrity and functional completeness of insect cuti-cle.The 2 key enzymes of tyrosine hydroxylase(TH)and dopa decarboxylase(DDC)predicted in melanin biosynthesis are usually conserved in insects.However,it is unclear whether their function is related to epidermal permeability.In this study,we identified and cloned the gene sequences of BgTH and BgDdc from Blattella germanica,and revealed that they both showed a high expression at the molting,and BgTH was abundant in the head and integument while BgDdc was expressed highest in the fat body.Using RNA in-terference(RNAi),we found that knockdown of BgTH caused molting obstacles in some cockroaches,with the survivors showing pale color and softer integuments,while knock-down of BgDdc was viable and generated an abnormal light brown body color.Desiccation assay showed that the dsBgTH-injected adults died earlier than control groups under a dry atmosphere,but dsBgDdc-injected cockroaches did not.In contrast,when dsRNA-treated cockroaches were reared under a high humidity condition,almost no cockroaches died in all treatments.Furthermore,with eosin Y staining assay,we found that BgTH-RNAi resulted in a higher cuticular permeability,and BgDdc-RNAi also caused slight dye pen-etration.These results demonstrate that BgTH and BgDdc function in body pigmentation and affect the waterproofing ability of the cuticle,and the reduction of cuticular perme-ability may be achieved through cuticle melanization.展开更多
基金funded by the National Natural Science Foundation of China(21576134 and 21706126)the National Key Research and Development Program(2016YFA0204300)。
文摘Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.
基金Open access funding provided by Karolinska Institutesupported by Karin and Sten Mörtstedt CBD Solutions AB,the Swedish Parkinson fund,the ALF program of the Stockholm Stockholm City,Lexa/Nordstjernan,Knut and Alice Wallenberg Foundation,and Van Geest Foundation.PS is a Wallenberg Clinical Scholar.
文摘Background There is a need for biomarkers to support an accurate diagnosis of Parkinson’s disease(PD).Cerebrospinal fluid(CSF)has been a successful biofluid for finding neurodegenerative biomarkers,and modern highly sensitive multiplexing methods offer the possibility to perform discovery studies.Using a large-scale multiplex proximity extension assay(PEA)approach,we aimed to discover novel diagnostic protein biomarkers allowing accurate discrimination of PD from both controls and atypical Parkinsonian disorders(APD).Methods CSF from patients with PD,corticobasal syndrome(CBS),progressive supranuclear palsy(PSP),multiple system atrophy and controls,were analysed with Olink PEA panels.Three cohorts were used in this study,comprising 192,88 and 36 cases,respectively.All samples were run on the Cardiovascular II,Oncology II and Metabolism PEA panels.Results Our analysis revealed that 26 and 39 proteins were differentially expressed in the CSF of test and validation PD cohorts,respectively,compared to controls.Among them,6 proteins were changed in both cohorts.Midkine(MK)was increased in PD with the strongest effect size and results were validated with ELISA.Another most increased protein in PD,DOPA decarboxylase(DDC),which catalyses the decarboxylation of DOPA(L-3,4-dihydroxyphenylalanine)to dopamine,was strongly correlated with dopaminergic treatment.Moreover,Kallikrein 10 was specifically changed in APD compared with both PD and controls,but unchanged between PD and controls.Wnt inhibitory factor 1 was consistently downregulated in CBS and PSP patients in two independent cohorts.Conclusions Using the large-scale PEA approach,we have identified potential novel PD diagnostic biomarkers,most notably MK and DDC,in the CSF of PD patients.
文摘目的:通过对胃腺癌患者的腹腔灌洗液检测多巴脱羧酶(dopa decarboxylase D D C)m R N A表达,评估D D C成为预测胃腺癌腹膜微转移的新指标的可能性.方法:收集南昌大学第二附属医院胃肠外科87例胃腺癌患者的腹腔灌洗液,应用实时荧光定量聚合酶链反应(quantitative real time polymerase chain reaction,QRT-PCR方法相对定量检测并比较DDC m RNA的表达.12例非癌症患者腹腔灌洗液作为阴性对照.结果:87例胃腺癌患者中,T1有6例,T2有14例,T3有28例,T4有39例,DDC m RNA的表达在不同T分级(浸润深度分级)DDC m RNA的相对表达值(×107)分别为:T1,168±21T2,283±87;T3,31162±4261;T4,35310±6593.非癌症组腹腔灌洗液中,DDC m RNA相对表达值(×107)为:60.28±19.00.此外D D C的表达还与组织分化程度、病理分化类型、是否有淋巴结转移等有关系.胃腺癌腹腔灌洗液应用常规腹腔细胞学检查(conventional intraperitoneal cytology,CY)检查,11例阳性(CY+),阳性率为13%(11/87).11例(CY+)中有9例DDC m RNA表达相对值结果高于临界值,归类于DDC+,DDC的敏感性为86%(9/11),此外,在10例T1患者和14例T2患者中DDC+为2例,且非癌症组中均未见DDC+,DDC的特异性为92%(22/24).表明腹腔灌洗液中DDC在胃腺癌不同浸润深度下差异性表达(P<0.05),具有较好的敏感性和特异性.结论:应用QRT-PCR技术可以有效检测腹腔灌洗液中DDC m RNA表达,DDC可能成为预测胃癌腹膜转移的可靠指标.
基金supported by the Crafoord Foundationthe Lundbeck Foundationthe Danish Medical Research Council
文摘Monoamine neurotransmitters play an important role in the modulation of sensory, motor and autonomic functions in the spinal cord. Although traditionally it is believed that in mammalian spinal cord, monoamine neurotransmitters mainly originate from the brain, accumulating evidence indicates that especially when the spinal cord is injured, they can also be produced in the spinal cord. In this review, I will present evidence for a possible pathway for two-step synthesis of dopamine and serotonin in the spinal cord. Published data from different sources and unpublished data from my own ongoing projects indicate that monoenzymatic cells expressing aromatic L-amino acid decarboxylase（AADC）, tyrosine hydroxylase（TH） or tryptophan hydroxylase（TPH） are present in the spinal cord and that these TH and THP cells often lie in close proximity to AADC cells. Prompted by the above evidence, I hypothesize that dopamine and serotonin could be synthesized sequentially in two monoenzymatic cells in the spinal cord via a TH-AADC and a TPH-AADC cascade respectively. The monoamines synthesized through this pathway may compensate for lost neurotransmitters following spinal cord injury and also may play specific roles in the recovery of sensory, motor and autonomic functions.
文摘The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes （TH and DDC）, in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.
基金supported by the National Natural Science Foundation of China(grant no.31772533)。
文摘Melanin is involved in cuticle pigmentation and sclerotization of insects,which is critical for maintaining structural integrity and functional completeness of insect cuti-cle.The 2 key enzymes of tyrosine hydroxylase(TH)and dopa decarboxylase(DDC)predicted in melanin biosynthesis are usually conserved in insects.However,it is unclear whether their function is related to epidermal permeability.In this study,we identified and cloned the gene sequences of BgTH and BgDdc from Blattella germanica,and revealed that they both showed a high expression at the molting,and BgTH was abundant in the head and integument while BgDdc was expressed highest in the fat body.Using RNA in-terference(RNAi),we found that knockdown of BgTH caused molting obstacles in some cockroaches,with the survivors showing pale color and softer integuments,while knock-down of BgDdc was viable and generated an abnormal light brown body color.Desiccation assay showed that the dsBgTH-injected adults died earlier than control groups under a dry atmosphere,but dsBgDdc-injected cockroaches did not.In contrast,when dsRNA-treated cockroaches were reared under a high humidity condition,almost no cockroaches died in all treatments.Furthermore,with eosin Y staining assay,we found that BgTH-RNAi resulted in a higher cuticular permeability,and BgDdc-RNAi also caused slight dye pen-etration.These results demonstrate that BgTH and BgDdc function in body pigmentation and affect the waterproofing ability of the cuticle,and the reduction of cuticular perme-ability may be achieved through cuticle melanization.
