Investigation of extracellular L-citrulline concentration in the striatum during alcohol withdrawal in rats

In this study, changes in striatal extracellular L-citrulline concentrations were investigated hourly for 5 h following alcohol withdrawal in chronic alcohol feeding Wistar rats. Alcohol (7.2% ethyl alcohol, v/v) was given to rats as modified liquid diet for 20 days. Signs of alcohol withdrawal appeared from the 1st h of alcohol withdrawal and the total alcohol withdrawal scores remained higher during the course of experiments. The mean of basal levels of L-citrulline in the microdialysis samples collected in conscious rat model from the striatum of control and alcoholized rats were found to be 1.28 ± 0.48 M and 0.35 ± 0.08 M, respectively. L-citrulline levels in the striatum of alcoholized rats increased by 4 folds significantly within 1 h following alcohol withdrawal. The increased striatal L-citrulline concentration was blocked by N^G-nitro-L-arginine methyl ester (L-NAME; 60 mg/kg), a nitric oxide synthase inhibitor, pretreatment. Our results indicate an increased L-citrulline level in the rat striatum during early alcohol withdrawal and this situation may be related to an increased nitric oxide production.     

L-瓜氨酸对子痫前期中的氧化应激和滋养层细胞侵袭的调控机制研究

摘要 目的：研究L-瓜氨酸（L-Cit）对子痫前期（PE）大鼠模型的治疗作用,及其对氧化应激和滋养层细胞侵袭的影响。方法：建立N-硝基-L-精氨酸甲酯（L-NAME）诱导的PE大鼠模型后,将大鼠分为Control组、PE组、PE+低、中和高剂量L-Cit组（依次为L-L-Cit、M-L-Cit和H-L-Cit组,剂量依次为100、200和500 mg/kg）,n=6,连续给药7 d。在孕第21 d时,检测各组大鼠收缩压（SBP）、24 h尿蛋白、血清超氧化物歧化酶（SOD）和丙二醛（MDA）水平,并对胎盘组织进行苏木精伊红（HE）染色。将HTR-8/Svneo细胞分为Control组、缺氧/复氧（H/R）组、H/R+L-Cit组和H/R+L-Cit+sc-221593组,分别对细胞进行H/R处理,并使用L-Cit（200 μg/mL）和特异性ERK1/2抑制剂sc-221593（10 μmol/L）培养细胞48 h。通过Transwell测定细胞侵袭。通过Western blotting检测total-ERK1/2、p-ERK1/2、total-p38、p-p38、基质金属蛋白酶（MMP）-2和MMP-9的蛋白表达。结果：与PE组比较,L-L-Cit组、M-L-Cit组和H-L-Cit组的SBP和24 h尿蛋白水平均降低（P<0.05）;胎盘组织形态明显改善;血清SOD升高,MDA降低（P<0.05）;胎盘组织中ERK1/2和p38的磷酸化水平及MMP-2和MMP-9的蛋白表达水平均升高（P<0.05）。与H/R组比较,H/R+L-Cit组的侵袭细胞数量升高（P<0.05）;ERK1/2和p38的磷酸化水平及MMP-2和MMP-9的蛋白表达水平均升高（P<0.05）。与H/R+L-Cit组比较,H/R+L-Cit+sc-221593组的上述变化均被逆转（P<0.05）。结论：L-Cit可能通过激活ERK/JNK通路减轻PE中的氧化应激并促进滋养层细胞侵袭,从而减轻PE症状。     

重组钝齿棒杆菌全细胞转化生产L-瓜氨酸条件优化

实现了精氨酸脱亚胺酶(ADI)首次在钝齿棒杆菌Corynebacterium crenatum SYPA 5-5中的高效表达。通过Ni-NTA亲和层析纯化得到纯化ADI,经SDS-PAGE测定其分子量约为46.8 k Da,酶学性质研究发现ADI的最适催化温度为37℃,最适pH为6.5,ADI在最佳催化条件下作用于L-精氨酸的米氏常数为12.18 mmol/L,最大反应速率为0.36μmol/(min·mL)。优化了重组菌全细胞转化产L-瓜氨酸的工艺条件,在最优条件下可一次转化300 g/L L-精氨酸,转化速率达8 g/(L·h)。进行重组菌5 L罐发酵并进行罐上全细胞转化300 g/L L-精氨酸,一批菌体可进行多次转化,累计产量达1 900 g以上。     

Effects of L-citrulline oral supplementation on polymorphonuclear neutrophils oxidative burst and nitric oxide production after exercise

Seventeen volunteer male professional cyclists were randomly assigned to control or supplemented (6 g L-citrulline-malate) groups and participated in a cycling stage. Blood samples were taken in basal conditions, after the race and 3 h post-race. Citrulline supplementation significantly increased plasma concentration of both arginine and citrulline after the stage only in the supplemented group. Polymorphonuclear neutrophils (PMNs) from controls responded to exercise with a progressive decrease in ROS production. Supplemented PMNs significantly increased ROS production after exercise compared to basal values and diminished to values lower than basal at recovery. PMN nitrite concentration was significantly higher after exercise and recovery only in the supplemented group. Markers of oxidative damage—CK, LDH, malondialdehyde—and DNA damage remained unchanged in both groups. In conclusion, oral L-citrulline administration previous to a cycling stage increases plasma arginine availability for NO synthesis and PMNs priming for oxidative burst without oxidative damage.     

HD-8树脂分离天花粉中的L-瓜氨酸

本文研究了用HD8阳离子交换树脂从天花粉中分离L瓜氨酸的工艺条件。采用HD8阳离子交换树脂从天花粉水提取液中交换L瓜氨酸,该树脂对L瓜氨酸的静态交换容量为50.8mg·mL1。在流速为3BV·h1,提取液中L瓜氨酸浓度为9.64mg·mL1时,HD8树脂对L瓜氨酸的动态交换容量为46.8mg·mL1树脂,其动态平衡时间为12min。NH4OH洗脱HD8树脂载荷的L瓜氨酸效果好。控制洗脱液流速为5BV·h1,用8BV0.25mol·L1的NH4OH即可完全洗脱L瓜氨酸。采用HZ803大孔径吸附树脂吸附L瓜氨酸洗脱液中的色素,真空浓缩,并在pH=5.97、4℃条件下结晶L瓜氨酸,其收得率为7.24%,其中L瓜氨酸含量为82.7%。与等电点法相比,产品纯度提高2.36倍。     

精氨酸脱亚胺酶活性以及瓜氨酸转化研究

目前,L-瓜氨酸的主要生产方法为化学法水解L-精氨酸.利用精氨酸脱亚胺酶可以直接转化L-精氨酸获得L-瓜氨酸,条件温和、效率高、环境友好,因而在工业化生产中具有很好的应用前景.本研究将优化后的单增李斯特菌(Listeriamonocytogenes)精氨酸脱亚胺酶(ADI)基因序列分别克隆到相应的载体pET-21a、pWB980及pAO815中,并分别转化大肠杆菌BL21(DE3)、枯草芽胞杆菌WB600及毕赤酵母GS115中构建表达菌株.发现大肠杆菌能高效表达ADI.经过5L发酵罐验证,发酵后酶活力单位可达200 000～270 000 U·L-1.经过验证,该酶可以很好地应用于L-瓜氨酸生产.在7 920～17 600 U· L-1的ADI存在条件下,5.5h内,可使95％的浓度为94～258 g·L-1的精氨酸转化为瓜氨酸.该方法具备良好的工业化应用前景.     

口服L-瓜氨酸对大鼠勃起功能的影响

给予雄性SD大鼠口服不同剂量组的L-瓜氨酸8周后,检测电刺激大鼠阴茎海绵体神经海绵体内压(ICP)的变化;比色法检测血清和组织中的一氧化氮(NO)含量、一氧化氮合酶(NOS)活性及血清中超氧化物歧化酶(SOD)活性、谷胱甘肽(GSH)含量等。结果高剂量(4.5 g.kg-1)L-瓜氨酸组ICP、NO含量、NOS活性显著增高;低、高剂量组SOD活性、GSH含量变化差异均无显著性。     

Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.