微小 RNA-194-5p与 cullin4A在肺结核病人中的表达及意义

目的 探讨微小RNA-194-5p(miR-194-5p)与泛素连接酶cullin4A(CUL4A)在肺结核病人中的表达及其意义.方法 选取2016年12月至2018年12月中国平煤神马集团职业病防治院结核病病人为研究对象,确诊为肺结核病人50例作为肺结核组,潜伏结核感染病人50例作为潜伏结核感染组,同期该院体检健康者50例作为对照组.实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测miR-194-5p、CUL4A mRNA、鸟苷酸结合蛋白5(GBP5)mRNA的表达;酶联免疫吸附测定(ELISA)检测可溶性Tim-3(sTim-3)、可溶性Gal-9(sGal-9)水平;采用Pearson相关性分析检验肺结核病人miR-194-5p、CUL4A表达量与GBP5、sTim-3、sGal-9的相关性.结核分枝杆菌(Mtb)感染人巨噬细胞系U937,ELISA检测白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)水平.蛋白质印迹法(Western blotting)检测Wnt/β-连环素(Wnt/β-catenin)信号通路相关蛋白T细胞因子(TCF)、糖原合成酶激酶-3(GSK-3β)、β-catenin表达量.结果 与对照组相比,潜伏结核感染组与肺结核组病人血清miR-194-5p、GBP5 mRNA的表达水平升高(P<0.05),其中miR-194-5p:(1.01±0.23)比(2.32±0.53)比(3.35±0.20),sTim-3、sGal-9水平升高(P<0.05),CUL4A mRNA的表达水平显著降低[(1.03±0.11)比(0.73±0.12)比(0.41±0.15),P<0.05],潜伏结核感染组与肺结核组各指标间相比差异有统计学意义(P<0.05);miR-194-5p与CUL4A呈负相关(r=-0.421,P<0.001),miR-194-5p与GBP5、sTim-3、sGal-9呈正相关(P<0.05);Mtb感染细胞后,IL-6、TNF-α的表达水平升高(P<0.05),TCF、GSK-3β、β-catenin表达量升高(P<0.05).结论 miR-194-5p在肺结核病人中表达上调,CUL4A表达下调,二者可能参与肺结核发生及发展过程.     

长链非编码 RNA GAS5、微小 RNA-21在多囊卵巢综合征中的表达及生物学意义

目的 探讨长链非编码RNA(LncRNA)GAS5与微小RNA-21(miR-21)在多囊卵巢综合征(PCOS)中的表达及其对卵巢颗粒细胞增殖及凋亡的影响.方法 收集2017年4月至2018年10月于南阳市中心医院就诊的50例PCOS病人为观察组,选取同时期于南阳市中心医院就诊的45例非PCOS病人为对照组,实时荧光定量聚合酶链式反应(qRT-PCR)检测对照组与观察组病人血清中GAS5与miR-21的表达水平.采用Pearson法分析PCOS病人中GAS5与miR-21表达的相关性.原代培养大鼠卵巢颗粒细胞,分别将pcDNA、pcDNA-GAS5转染至卵泡颗粒细胞.CCK-8检测细胞增殖;流式细胞术检测细胞凋亡率;双荧光素酶报告实验验证GAS5与miR-21的靶向关系.结果 观察组病人血清中GAS5的表达水平明显低于对照组[(0.52±0.11)比(1.00±0.12),P<0.05],miR-21的表达水平明显高于对照组[(2.36±0.57)比(1.01±0.10),P<0.05],GAS5与miR-21呈负相关(r=?0.310,P=0.028);TT、IL-18与RGAS5呈负相关(P<0.05),而与miR-21呈正相关(P<0.05);与pcDNA组相比,pcDNA-GAS5组细胞活力降低(P<0.05),细胞凋亡率升高[(36.49±5.21)比(8.52±1.20),P<0.05];双荧光素酶报告实验证实GAS5可靶向结合miR-21,并可负向调控miR-21的表达.结论 GAS5过表达可能通过靶向调控miR-21表达从而抑制卵巢颗粒细胞增殖,促进细胞凋亡.     

血清长基因间非编码RNA00511、微RNA-515-5p在子宫内膜癌中表达与临床病理特征及预后的关系

目的 探究子宫内膜癌病人血清长基因间非编码RNA00511(LINC00511)、微RNA-515-5p(miR-515-5p)表达水平与临床病理特征及预后的关系。方法 收集2017年3月至2019年2月海南医学院第二附属医院子宫内膜癌病人（子宫内膜癌组）82例;同时收集子宫内膜不典型增生病人（不典型增生组）67例及健康体检者（对照组）79例。采用实时荧光定量逆转录聚合酶链式反应（qRT-PCR）检测血清LINC00511、miR-515-5p表达水平;采用Pearson相关分析子宫内膜癌病人血清LINC00511与miR-515-5p表达水平的相关性;采用Kaplan-Meier法分析子宫内膜癌病人血清LINC00511、miR-515-5p表达水平与预后的关系;多因素Cox回归分析影响子宫内膜癌病人预后的因素。结果 对照组、不典型增生组、子宫内膜癌组血清LINC00511表达水平（1.02±0.21、1.82±0.31、2.60±0.45）依次升高,miR-515-5p表达水平（1.01±0.20、0.70±0.18、0.42±0.16）依次降低（P<0.05）。肌层浸润深度...     

超声造影定量参数联合血清微小 RNA-139-5p、微小 RNA-15a检测在卵巢浆液性囊腺癌病人中应用价值

目的 分析超声造影定量参数联合血清微小RNA-139-5p(miR-139-5p)、微小RNA-15a(miR-15a)检测在卵巢浆液性囊腺癌病人中应用价值及意义.方法 选取2017年6月至2019年9月重庆市北碚区中医院收治的92例卵巢浆液性囊腺癌病人为腺癌组及92例卵巢浆液性囊腺瘤病人为腺瘤组.行超声造影检查与血清miR-139-5p、miR-15a水平检测,观察超声造影定量参数(始增时间、达峰时间、增强强度)、血清miR-139-5p、miR-15a水平两组间差异,多因素logistic回归分析各指标与卵巢浆液性囊腺癌的相关性,ROC曲线评估其对卵巢浆液性囊腺癌的诊断效能,Pearson相关分析各指标与淋巴结转移的关系.结果 腺癌组超声造影始增时间、达峰时间、血清miR-15a水平分别为(14.16±2.51)s、(22.28±4.64)s、(0.04±0.02),均低于腺瘤组的(17.48±3.09)s、(29.30±4.97)s、(0.11±0.06)(P<0.05),超声造影增强强度与血清miR-139-5p水平分别为(89.24±12.83)dB、(8.76±2.90),均高于腺瘤组的(73.38±10.74)dB、(4.09±2.52)(P<0.05);logistic回归分析显示,超声造影始增时间、达峰时间、增强强度、血清miR-139-5p、miR-15a均与卵巢浆液性囊腺癌具有相关性(P<0.05);ROC曲线显示,超声造影定量参数与血清指标联合诊断卵巢浆液性囊腺癌的AUC为0.896,高于各单一指标,其诊断敏感度为90.22％,特异度为78.26％;有淋巴结转移卵巢浆液性囊腺癌病人超声造影始增时间、达峰时间、血清miR-15a水平分别为(12.53±2.92)s、(19.84±3.69)s、(0.02±0.01),均低于无淋巴结转移病人的(15.59±2.24)s、(24.42±4.87)s、(0.06±0.03),超声造影增强强度、血清miR-139-5p水平分别为(97.35±14.06)dB、(10.14±3.08),均高于无淋巴结转移病人(82.12±11.63)dB、(7.55±2.86)(P<0.05);Pearson相关性分析显示超声造影始增时间、达峰时间及血清miR-15a水平与卵巢浆液性囊腺癌病人淋巴结转移呈负相关,超声造影增强强度及血清miR-139-5p水平与卵巢浆液性囊腺癌病人淋巴结转移呈正相关(P<0.05).结论 应用超声造影定量参数联合血清miR-139-5p、miR-15a检测可显著提升卵巢浆液性囊腺癌早期诊断效能,且其水平与淋巴结转移密切相关,可为临床诊治提供可靠依据.     

miR-21-5p在急性有机磷农药中毒患者血清中的表达意义及与CK-MB、AMS水平的相关性分析

目的探究miR-21-5p在急性有机磷农药中毒患者血清中的表达意义及与肌酸磷化脢-同功脢MB(CK-MB)、血清淀粉酶(AMS)水平的相关性。方法选择2018年9月至2019年12月在我院救治的60例急性有机磷农药中毒患者作为中毒组,50例正常体检者作为对照组。入院后采取患者静脉血3 ml,qPCR检测急性有机磷农药中毒患者血清miR-21-5p表达水平,ELISA法检测急性有机磷农药中毒患者血清CK-MB、AMS水平。分析两组间血清miR-21-5p表达水平的差异及其与CK-MB、AMS水平的相关性。结果qRT-PCR结果显示,中毒组血清miR-21-5p相对表达量为1.48±0.36,明显高于对照组的1.05±0.31,差异有统计学意义(P<0.05)。ELISA结果显示,中毒组血清CK-MB和AMS相对表达量明显高于对照组的相对表达量,差异均有统计学意义(P<0.05)。血清miR-21-5p与CK-MB和AMS水平呈正相关性(P<0.05)。血清miR-21-5p的表达与患者是否有呼吸衰竭和胰腺炎有关(P<0.05)。结论miR-21-5p在急性有机磷农药中毒患者血清中的表达显著升高且与CK-MB、AMS水平呈正相关。     

非小细胞肺癌患者血清LncRNA FAM138B、miR-105-5p表达与病理特征的关系

目的 探讨非小细胞肺癌(NSCLC)患者血清长链非编码RNA序列相似性家族138成员B(LncRNA FAM138B)、微小RNA-105-5p(miR-105-5p)表达与病理特征的预后的关系。方法 选取2018年1月至2019年12月收治的110例NSCLC患者为NSCLC组,另选取同期60例体检健康者为对照组,采用实时荧光定量聚合酶链式反应检测血清LncRNA FAM138B、miR-105-5p表达。分析血清LncRNA FAM138B、miR-105-5p表达与NSCLC患者病理特征的关系。StarBase数据库预测LncRNA FAM138B与miR-105-5p的关系。采用Pearson相关性分析NSCLC患者血清LncRNA FAM138B与miR-105-5p表达的相关性,多因素Cox回归分析NSCLC患者预后影响因素,受试者工作特征(ROC)曲线分析血清LncRNA FAM138B、miR-105-5p表达对NSCLC患者预后的预测价值。结果 与对照组比较,NSCLC组血清LncRNA FAM138B表达降低,miR-105-5p表达升高(P<0.05)。经S...     

微小 RNA361-5p在肺结核病人血清中水平及意义

目的 探究微小RNA361-5p(miR-361-5p)在肺结核病人血清中水平及意义.方法 选取2017年3月至2019年1月开封市结核病防治所诊治的肺结核病人136例为肺结核组;并选择同时间段内同一医院142例健康体检者为健康对照组.比较两组一般临床资料;以实时荧光定量PCR(qRT-PCR)法检测两组血清miR-361-5p水平;检测血管内皮生长因子(VEGF)、肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)水平;分析肺结核病人血清miR-361-5p水平与VEGF、TNF-α、IFN-γ的关系;回归分析肺结核发生的影响因素;分析血清miR-361-5p对肺结核的诊断价值.结果 肺结核组血清miR-361-5p[(1.58±0.45)比(1.06±0.31)]、VEGF[(389.39±46.85)pg/mL比(327.62±41.76)pg/mL]、TNF-α[(22.48±5.06)ng/L比(11.36±3.17)ng/L]水平均明显高于健康对照组,IFN-γ水平[(15.58±3.36)ng/L比(26.72±4.78)ng/L]明显低于健康对照组(均P<0.05);肺结核病人血清miR-361-5p与VEGF、TNF-α水平呈正相关,与IFN-γ水平呈负相关(均P<0.05);miR-361-5p、VEGF、TNF-α是影响肺结核发生的危险因素,IFN-γ是影响肺结核发生的保护因素(均P<0.05);血清miR-361-5p水平对肺结核发生诊断的AUC为0.898,截断值为1.44,其灵敏度为83.8％,特异度为88.0％.结论 肺结核病人血清miR-361-5p呈高表达,与炎症相关因子密切相关,miR-361-5p可能与炎症相关因子相互作用,进而共同调节肺结核发生、发展.     

自身免疫性甲状腺炎甲状腺组织和外周血单个核细胞中微小 RNA-142-3p、微小 RNA-150表达及临床意义

目的 探讨甲状腺组织和外周血单个核细胞中微小RNA-142-3p(miR-142-3p)、微小RNA-150(miR-150)在桥本甲状腺炎(HT)中的表达水平及临床意义.方法 选取2017年3月至2019年6月南阳市中心医院行甲状腺手术切除并经病理确诊为HT病人87例为HT组;同期选取南阳市中心医院行甲状腺腺瘤切除术的非癌病人73例为对照组.采用实时荧光定量PCR(qRT-PCR)法检测两组甲状腺组织和外周血单个核细胞中miR-142-3p、miR-150表达水平;分析HF病人miR-142-3p、miR-150水平与甲状腺功能参数及相关细胞因子相关性.结果 HT组甲状腺组织和外周血单个核细胞中miR-142-3p[(1.54±0.32)、(2.25±0.54)]比[(1.00±0.23)、(1.00±0.24)]、miR-150[(2.21±0.43)、(3.78±1.14)]比[(1.00±0.16)、(1.00±0.19)]表达水平均显著高于对照组(P<0.05);两组甲状腺功能参数及相关细胞因子水平比较,均差异有统计学意义,HT组促甲状腺激素(TSH)、甲状腺过氧化物酶抗体(TPOAb)、白细胞介素17(IL-17)、白细胞介素6(IL-6)、干扰素-γ(IFN-γ)、转化生长因子β1(TGF-β1)水平显著高于对照组(P<0.05),游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)水平显著低于对照组(P<0.05);HT病人甲状腺组织和外周血单个核细胞中miR-142-3p、miR-150水平呈正相关,且与TSH、TPOAb、IL-17、IL-6、IFN-γ、TGF-β1呈正相关,与FT3、FT4呈负相关(P<0.05).结论 甲状腺组织和外周血单个核细胞中miR-142-3p、miR-150水平的变化与HT有关.miR-142-3p、miR-150对评价HT的早期危险性、降低患病率有重要意义.     

术前血清miR-483-5p水平在肝内胆管细胞癌诊断及预后中的价值

目的:探讨术前血清miR-483-5p水平在肝内胆管细胞癌(ICC)早期诊断及预后评估中的价值。方法:选择2015年12月—2017年12月收治的91例ICC患者为ICC组,选择同期40例健康体检者为对照组,统计一般临床资料并检测血清miR-483-5p水平,采用受试者工作特征曲线,分析miR-483-5p对ICC的诊断效能。根据miR-483-5p中位值将ICC患者分为低表达组和高表达组,分析比较两组患者病理特征的差异,应用Kaplan-Meier法进行生存分析,并对生存期进行单因素和多因素Cox回归分析。结果:ICC组患者血清miR-483-5p水平明显高于对照组(P<0.05);miR-483-5p诊断ICC的曲线下面积为0.758,敏感性55.00%,特异性86.81%。高表达组和低表达组之间的肿瘤分化程度和淋巴结转移比较,差异有统计学意义(P<0.05)。Kaplan-Meier生存分析显示,高表达组患者的整体生存期明显低于低表达组(P<0.05)。单因素及多因素Cox回归分析发现,血清miR-483-5p水平与ICC患者术后生存期有关,但不是生存期的独立影响因素。结论:血清miR-483-5p水平对ICC具有一定诊断价值,且高miR-483-5p水平ICC患者的术后生存期相对较短,可作为ICC早期诊断及预后评估辅助评价指标。     

子宫内膜癌患者血清和癌组织中LncRNA CRNDE、miR-153-5p表达及临床诊断价值

目的 探讨miR-153-5p、LncRNA CRNDE在子宫内膜癌(EC)患者血清和癌组织中的表达及对EC的诊断价值。方法 选取2019年12月至2021年12月手术治疗的115例EC患者为观察组，收集手术患者的癌组织及癌旁组织，同时选取100例体检健康者为对照组。实时荧光定量PCR(qRT-PCR)法检测血清和癌组织中miR-153-5p、LncRNA CRNDE表达水平，分析血清中miR-153-5p、LncRNA CRNDE表达水平与患者临床病理参数的关系；受试者工作特征(ROC)曲线评价血清LncRNA CRNDE、miR-153-5p表达水平对EC发生的诊断价值。结果 与癌旁组织相比，LncRNA CRNDE在癌组织中的表达水平升高，miR-153-5p表达水平降低，且二者呈负相关(r=-0.268,P<0.05);与对照组比较，LncRNA CRNDE在观察组患者血清中的表达水平升高，miR-153-5p表达水平降低，且二者呈负相关(r=-0.315,P<0.05)。EC患者血清中LncRNA CRNDE、miR-153-5p表达水平均与年龄、肿瘤直径、组织类...     

Serotonin synthesis with rat brain synaptosomes. Effects of L-dopa, L-3-methoxytyrosine and catecholamines

Experiments were done with a fraction of rat brain containing mitochondria and synaptosomes. L-DOPA∥ (K_t 0·3 mM) and L-3-methoxytyrosine (K_t 0·5 mM) were found to be competitive inhibitors of tryptophan accumulation, while dopamine and l-noradrenaline had no effect on the accumulation of tryptophan. Furthermore, L-DOPA (K_t 5·6 μM) was about 100 times more potent than L-3-methoxytyrosine in inhibiting the synthesis of 5HT from tryptophan. The inhibition of 5HT synthesis by L-DOPA appeared to be competitive to tryptophan and was not linear at concentrations of L-DOPA higher than 1 μM. L-DOPA also interfered with the rate of deamination of 5HT synthesized in vitro. Dopamine and l-noradrenaline also inhibited the synthesis of 5HT.     

Mechanism of block by fluoxetine of 5-hydroxytryptamine3 (5-HT3)-mediated currents in NCB-20 neuroblastoma cells

The effect of fluoxetine (Prozac) on 5-hydroxytryptamine(3) (5-HT(3))-mediated currents in NCB-20 neuroblastoma cells was examined using the whole-cell patch-clamp technique. Fluoxetine produced a significant reduction of peak amplitude without altering the activation time course of 5-HT(3)-mediated currents. These effects were concentration-dependent, with an IC(50) value of 4.15 microM. No voltage dependence was evident in fluoxetine's block of 5-HT(3)-mediated currents over the entire voltage range tested. The extent of block by pre-application of fluoxetine was significantly greater than that by co-application. Fluoxetine also increased the apparent rate of current desensitization to 5-HT application. Using a first-order kinetics analysis, the open-channel blocking rate constants were 0.06 microM(-1)s(-1) (k(+1), association rate constant) and 0.05 s(-1) (k(-1), dissociation rate constant), with an apparent K(d) (=k(-1)/k(+1)) of 0.83 microM. This value is close to an IC(50) of 1.11 microM obtained from the reduction in tau, the time constant of desensitization. Intracellular application of fluoxetine for long durations had no effect on the amplitude or kinetics of 5-HT(3)-mediated currents. Similarly, norfluoxetine, the major metabolite of fluoxetine, reduced the peak current, and enhanced the rate of current desensitization in a concentration-dependent manner with an IC(50) of 2.66 microM, indicating that norfluoxetine is more potent than fluoxetine in blocking 5-HT(3)-mediated currents. These results indicate that, at clinically relevant concentrations, fluoxetine and its metabolite, norfluoxetine, block 5-HT(3)-mediated currents in NCB-20 neuroblastoma cells.     

Kinetic studies with 5-azacytidine-5'-triphosphate and DNA-dependent RNA polymerase.

In order to further understand the biochemical mode of action of 5-azacytidine, a potent antileukemic agent, kinetic studies were performed with 5-azacytidine-5'-triphosphate (5-aza-CTP) and purified DNA-dependent RNA polymerase from Escherichia coli and calf thymus. RNA polymerase could catalyze the incorporation of the fradulent nucleotide, 5-aza-CTP, into RNA. The apparent K_m value for 5-aza-CTP was estimated to be 350 and 390 for the E. coli and calf thymus enzymes respectively. The K_m value for 5-aza-CTP was about 18-fold greater than the K_m value for CTP (20 μM). The apparent V_max value for CTP was about 2-fold greater than the V_max value for 5-aza-CTP. 5-Aza-CTP was a weak competitive inhibitor with respect to CTP; the apparent K_i value for 5-aza-CTP was estimated to be 680 and 810 μM for the E. coli and calf thymus enzymes respectively. On the other hand, CTP was a potent competitive inhibitor with respect to 5-aza-CTP; the apparent K_i value of CTP was estimated to be 16 μM. 5-Aza-CTP did not appear to inhibit the incorporation of UTP into RNA in the reaction catalyzed by RNA polymerase. These data suggest that the inhibition of RNA synthesis in cells by 5-aza-cytidine is not produced by the inhibition of RNA polymerase by 5-aza-CTP.     

Effect of clonidine on the 5-hydroxytryptamine and 5-hydroxyindoleacetic acid brain levels

The effects of clonidine on the brain levels of 5-HT and 5-HIAA in rats and mice were studied. Clonidine did not change the levels of 5-HT and 5-HIAA in the whole brains of either animal species but the 5-HT concentrations were elevated in rat brain pons + medulla oblongata. Clonidine antagonized the increase in the brain 5-HIAA levels induced by apomorphine in rats and mice. The decrease in the 5-HT level and the increase in the 5-HIAA level observed in rats after L-dopa (given with peripheral decarboxylase inhibitor RO 4-4602) were counteracted by clonidine.     

Effect of de novo purine synthesis inhibitors on 5-fluorouracil metabolism and cytotoxicity

Methotrexate pretreatment of L1210 cells had been shown previously by us to cause an enhancement of the intracellular accumulation of 5-fluorouracil and of the formation of 5-fluorouracil nucleotides which was correlated with synergistic cytotoxicity. This effect of methotrexate was associated with increases in 5-phosphoribosyl-1-pyrophosphate, the cofactor required for the conversion of 5-fluorouracil to 5-fluorouridine-5'-monophosphate (FUMP). Because these influences on 5-fluorouracil metabolism were most likely mediated by the activity of methotrexate as an inhibitor of purine synthesis, the effects of other agents that inhibit purine synthesis were examined. An inhibitor of amidophosphoribosyltransferase, 6-methylmercaptopurine ribonucleoside, the glutamine antagonists, azaserine and 6-diazo-5-oxo-L-norleucine (DON), and the L-aspartate analogue inhibitor of adenylsuccinate synthetase, L-alanosine, all reduced the incorporation of [1-14C]glycine into adenine and guanine bases isolated from nucleic acids. Each drug also resulted in intracellular elevations of 5-phosphoribosyl-1-pyrophosphate that were 15- to 25-fold greater than control levels. These alterations in de novo purine nucleotide synthesis were associated with enhanced intracellular 5-fluorouracil accumulation and synergistic cytotoxicity.     

Exclusion of exogenous 5-methyl-2'-deoxycytidine from DNA in human leukemic cells. A study with [2(-14)C]- and [methyl-14C]5-methyl-2'-deoxycytidine

Modification of DNA-cytosine by a 5-methyl group is thought to be an important mechanism which regulates the expression of eukaryotic genes. This modification takes place after semiconservative replication. There is very little evidence, if any, that 5MeCyt could be naturally incorporated into mammalian DNA in semiconservative replication. We have clarified the possibility of incorporating 5MedCyd pharmacologically into human leukemic cells in vitro. To this end, we developed a novel small-scale synthesis method for 14C-labeled 5MedCyd starting from commercially available [14C]dThd derivatives. Particular attention was focused upon possible incorporation of radioactive 5MedCyd derivatives into the acid-soluble cellular fraction as well as into nucleic acids and protein in human cells. The results showed that [2(-14)C]- and [methyl-14C]5MedCyd were incorporated into human leukemic cells to a similar extent. The radioactivity originating from these compounds was incorporated mainly into the acid-soluble pool and nucleic acids. The exact nature of the intracellular radioactive molecules in RNA is not known, but the radioactive label in DNA hydrolyzate co-chromatographed exclusively with thymine. Hence, 5MedCyd is deaminated to thymidine before incorporating into DNA. This deamination had taken place already (partially) in the culture medium. Human leukemic cells do effectively protect their DNA from incorporation of exogenous 5MedCyd.     

Prenatal nicotine exposure in rhesus monkeys compromises development of brainstem and cardiac monoamine pathways involved in perinatal adaptation and sudden infant death syndrome: amelioration by vitamin C

Maternal smoking during pregnancy greatly enhances perinatal morbidity/mortality and is the major risk factor for Sudden Infant Death Syndrome (SIDS). Studies in developing rodents indicate that nicotine is a neuroteratogen that targets monoamine pathways involved in the responses to hypoxia that are in turn, hypothesized to contribute to these adverse events. We administered nicotine to pregnant Rhesus monkeys from gestational day 30 through 160 by continuous infusion, achieving maternal plasma levels comparable to those in smokers; we examined neurochemical parameters immediately after Cesarean delivery at the end of the exposure period. Nicotine evoked elevations in brainstem serotonin levels and serotonin turnover, indicating hyperactivity of these pathways. The same treatment evoked a deficit in cardiac norepinephrine levels. Both effects were offset by coadministration of the antioxidant, Vitamin C. Brainstem serotonin hyperinnervation is a hallmark of SIDS, and the hyperactivity seen here can also account for the downregulation of serotonin receptors noted in this disorder. Deficient cardiac sympathetic innervation is also consistent with increased vulnerability to hypoxia during delivery or in the agonal event in SIDS. Our results thus indicate that nicotine exposure in a primate model produces brainstem and autonomic abnormalities of the key monoamine systems that govern the response to hypoxia, indicate an important role of oxidative stress in the adverse effects, and point to potential amelioration strategies that could offset these particular effects of nicotine.     

Cytidine and deoxycytidylate deaminase inhibition by uridine analogs

Cytidine deaminase, an enzyme found in the supernatant fluid of hepatocytes, granulocytes and tumor cells, and in plasma, degrades the antitumor agents cytosine arabinoside and 5-azacytidine. Uridine and its analogs, 3-deazauridine, 5-bromodeoxyuridine, 5-fluorodeoxyuridine and 6-azauridine, were found to competitively inhibit cytidine deaminase; the most potent inhibitor was 3-deazauridine (K_i = 1.9 × 10^?5 M). In addition, deoxycytidylate deaminase, which degrades cytosine arabinoside monophosphate to the inactive uracil arabinoside monophosphate (K_m = 9 × 10^?4 M), was competitively inhibited by 3-deazauridine monophosphate, as well as by the nucleotides of other uridine analogs. These results suggest that uridine analogs such as 3-deazauridine may have value in protecting cytosine arabinoside, 5-azacytidine and their monophosphate nucleotides from degration by neucleoside and nucleotide deaminases.     

Analytical and pharmacokinetic studies with 5-chloro-2'-deoxycytidine

5-Chloro-2'-deoxycytidine (NSC 371331, CDC) is in development as a possible radiosensitizing agent for cancer treatment. Previous studies have been done to demonstrate the in vivo efficacy of CDC with various modulators of its metabolism. This paper describes our preclinical studies to determine the pharmacokinetic properties of CDC and the disposition of the drug, both alone and in the presence of the metabolic modulator tetrahydrouridine (THU), a cytidine deaminase inhibitor. Detection of the drug in biological fluids was performed by HPLC analysis using a C-18 column, gradient elution with solvents composed of aqueous trifluoroacetic acid and acetonitrile, and ultraviolet absorbance at 290 nm. Samples were processed by treatment with ammonium sulfate prior to injection into the HPLC system. CDC was stable in aqueous solution and in mouse plasma. High doses of CDC (100mg/kg) were given i.v. or i.p. to mice for the determination of CDC plasma half-life (10 min). CDC was not detectable in plasma after oral administration. It was converted rapidly to 5-chloro-2'-deoxyuridine (CDU) by cytidine deaminase, and CDU was readily discernable in plasma and urine samples collected after i.v. and i.p. administration of CDC. When CDC in doses ranging from 5 to 100mg/kg was given with 100mg/kg of THU, increased plasma levels of CDC were seen. CDC was eliminated through the kidneys, as well as by enzymatic deamination, and did not bind to plasma proteins. The initial steps of the CDC metabolic pathway were determined in vitro with isolated enzymes. Cytidine deaminase from mouse kidney converted CDC into CDU; thymidine phosphorylase converted CDU into 5-chlorouracil (5-CU). The conclusions of these studies are: (a) CDC is a drug with a short half-life and (b) it is excreted through the kidney, mainly in metabolite form. Administration of THU substantially increased the concentrations of CDC in mouse plasma, supporting proposals that the combination of THU with CDC should be evaluated in clinical trials.     

Aromatic L-amino acid decarboxylase in calcitonin-producing cells

A common feature of the calcitonin-producing cells (C cells) is their capacity to produce and store arylethylamines. The activity of aromatic l-amino acid decarboxylase (DOPA/5-HTP decarboxylase) was measured radiometrically in the thyroid glands of various species and in the ultimobranchial gland of the chicken. The enzyme activity was well correlated with the number of amine-containing C cells, demonstrated by fluorescence microscopy in these tissues. The ultimobranchial gland had a conspicuously high activity of aromatic amino acid decarboxylase. The follicular cells of the thyroid appeared to have no or only a very low activity of this enzyme.