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
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
Insect immune-associated phospholipase A_(2) (PLA_(2)) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate i...Insect immune-associated phospholipase A_(2) (PLA_(2)) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate immune attack against invaders. However, the effect of PLA_(2) on melanization has not yet been reported in insects or other arthropods. In this work, we cloned a PLA_(2) gene (BmsPLA_(2)), and its protein had characteristic features of secreted PLA_(2) (sPLA_(2)). After injection of bacteria, BmsPLA_(2) expression and sPLA_(2) activity in hemolymph significantly increased. BmsPLA_(2) fluorescence was transferred from the cytoplasm to the cell membranes of circulating hemocytes. These results indicated that BmsPLA_(2) was related to hemolymph immunity in silkworms. Interestingly, reducing BmsPLA_(2) by RNA interference decreased melanosis (melanistic hemocytes) levels in vivo and in vitro, while BmsPLA_(2) overexpression had the opposite effect. The larval survival and melanization rate in the hemocoel both slowed depending on the PLA_(2) inhibitor dosage. These results demonstrated that BmsPLA_(2) plays a role in melanization during the immune process of silkworms. Surprisingly, the level of BmDDC matched the degree of melanization in various observations. BmDDC expression showed a significant increase, with the peak occurring later than that of BmsPLA_(2) after injection of bacteria, implying that BmsPLA_(2) was activated prior to BmDDC. Moreover, the alteration of BmsPLA_(2) by RNA interference or overexpression led to altered BmDDC levels. These results suggested that BmsPLA_(2) regulates the melanization response in silkworms through BmDDC. Our study proposes a new regulatory mechanism of the melanization response and new directions for understanding the complex immune networks of insects.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.
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.
基金supported by the Basic research and frontier exploration projects of Chongqing(grant num-bers cstc2018jcyjAX0075)the Subsidy fund for the development of National Silk in Chongqing(grant number 20210607223136209)the Natural Science Foundation Unode Program of Chongqing(grant number CSTS2013jjB80004).
文摘Insect immune-associated phospholipase A_(2) (PLA_(2)) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate immune attack against invaders. However, the effect of PLA_(2) on melanization has not yet been reported in insects or other arthropods. In this work, we cloned a PLA_(2) gene (BmsPLA_(2)), and its protein had characteristic features of secreted PLA_(2) (sPLA_(2)). After injection of bacteria, BmsPLA_(2) expression and sPLA_(2) activity in hemolymph significantly increased. BmsPLA_(2) fluorescence was transferred from the cytoplasm to the cell membranes of circulating hemocytes. These results indicated that BmsPLA_(2) was related to hemolymph immunity in silkworms. Interestingly, reducing BmsPLA_(2) by RNA interference decreased melanosis (melanistic hemocytes) levels in vivo and in vitro, while BmsPLA_(2) overexpression had the opposite effect. The larval survival and melanization rate in the hemocoel both slowed depending on the PLA_(2) inhibitor dosage. These results demonstrated that BmsPLA_(2) plays a role in melanization during the immune process of silkworms. Surprisingly, the level of BmDDC matched the degree of melanization in various observations. BmDDC expression showed a significant increase, with the peak occurring later than that of BmsPLA_(2) after injection of bacteria, implying that BmsPLA_(2) was activated prior to BmDDC. Moreover, the alteration of BmsPLA_(2) by RNA interference or overexpression led to altered BmDDC levels. These results suggested that BmsPLA_(2) regulates the melanization response in silkworms through BmDDC. Our study proposes a new regulatory mechanism of the melanization response and new directions for understanding the complex immune networks of insects.
