不明原因复发性流产再次妊娠患者血清1,25(OH)2D3、sTim-3与Th17/Treg免疫失衡和妊娠结局的关系

摘要 目的：探讨不明原因复发性流产（URSA）再次妊娠患者血清1,25-二羟维生素D3[1,25（OH)）₂D₃]、可溶性T细胞免疫球蛋白黏蛋白分子3（sTim-3）与辅助性T细胞17（Th17）/调节性T细胞（Treg）免疫失衡和妊娠结局的关系。方法：选择于湖南省妇幼保健院2020年1月~2022年1月就诊的62例URSA再次妊娠患者作为研究组,另选择同期进行孕检的正常早孕妇女30例作为对照组。比较两组孕早期血清1,25(OH) ₂D₃、sTim-3及外周血Th17细胞、Treg细胞水平、Th17/Treg比值。Pearson法分析URSA再次妊娠患者血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值平的相关性。根据URSA再次妊娠患者妊娠结局的不同分为妊娠成功分娩组和妊娠再次流产组,比较两组孕早期血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值。受试者工作特征（ROC）曲线分析血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值对妊娠结局的预测价值。结果：研究组血清sTim-3、外周血Th17细胞水平、Th17/Treg比值高于对照组,血清1,25(OH) ₂D₃、外周血Treg细胞水平低于对照组（P<0.05）。Pearson相关分析显示,URSA再次妊娠患者血清1,25(OH) ₂D₃与血清sTim-3、外周血Th17细胞水平、Th17/Treg比值呈负相关,与Treg细胞水平呈正相关（P<0.05）;血清sTim-3与外周血Treg细胞水平呈负相关,与Th17细胞水平、Th17/Treg比值呈正相关（P<0.05）。妊娠再次流产组血清sTim-3、外周血Th17细胞水平、Th17/Treg比值高于妊娠成功分娩组,血清1,25(OH) ₂D₃、外周血Treg细胞水平低于妊娠成功分娩组（P<0.05）。ROC曲线分析显示,血清1,25(OH) ₂D₃、sTim-3及外周血Th17细胞、Treg细胞水平及Th17/Treg比值均可预测URSA再次妊娠患者妊娠再次流产的发生风险,且上述指标联合检测的预测效能更高。结论：血清1,25(OH) ₂D₃水平异常降低、sTim-3水平异常升高可导致Th17/Treg免疫失衡,导致URSA再次妊娠患者再次发生流产。上述指标联合检测对URSA再次妊娠患者妊娠再次流产的预测效能更高。     

慢性牙周炎患者血清CGRP、PGE2、CCL20与牙周临床指标和Th17/Treg失衡的相关性分析

摘要 目的：探讨慢性牙周炎（CP）患者血清降钙素基因相关肽（CGRP）、前列腺素E₂（PGE₂）、C-C基序趋化因子配体20（CCL20）与牙周临床指标和辅助性T细胞17/调节性T细胞（Th17/Treg）失衡的相关性。方法：选取2018年3月～2022年1月本院收治的92例CP患者,根据病情严重程度分为轻度组28例、中度组33例、重度组31例。检查患者牙周临床指标,采用酶联免疫吸附法检测血清CGRP、PGE₂、CCL20水平,流式细胞术检测外周血Th17、Treg百分比并计算Th17/Treg。比较3组血清CGRP、PGE₂、CCL20水平和牙周临床指标及外周血Th17、Treg、Th17/Treg,采用Pearson相关系数分析CP患者血清CGRP、PGE₂、CCL20水平与牙周临床指标和Th17/Treg的相关性。结果：轻度组、中度组、重度组血清CGRP水平和外周血Treg比例依次降低,血清PGE₂、CCL20水平和探诊深度（PD）、出血指数（BI）、附着丧失（AL）、菌斑指数（PLI）及外周血Th17比例、Th17/Treg依次升高（P＜0.05）。Pearson相关系数显示,CP患者血清CGRP水平与PD、BI、AL、PLI、Th17/Treg呈负相关,PGE₂、CCL20水平与PD、BI、AL、PLI、Th17/Treg呈正相关（P＜0.05）。结论：血清CGRP、PGE₂、CCL20水平与CP患者牙周临床指标和Th17/Treg失衡密切相关,可能是CP发生发展的关键因子。     

龈沟液miR-155、miR-223表达水平与慢性牙周炎伴2型糖尿病患者牙周临床指标、口腔龈下菌群以及Th17/Treg失衡的相关性分析

摘要 目的：探讨龈沟液miR-155、miR-223表达水平与慢性牙周炎伴2型糖尿病（T2DM）患者牙周临床指标、口腔龈下菌群以及外周血辅助性T细胞17（Th17）/调节性T细胞（Treg）失衡的相关性。方法：选择2018年1月至2022年1月安徽理工大学第一附属医院口腔科收治的86例慢性牙周炎患者,根据是否伴T2DM将患者分为慢性牙周炎伴T2DM组15例和单纯慢性牙周炎组71例,另选择65例健康体检志愿者为对照组。检测龈沟液miR-155、miR-223表达水平,口腔龈下菌群以及外周血Th17细胞占比、Treg细胞占比、血清白细胞介素（IL）-17、转化生长因子-β（TGF-β）水平。Pearson相关分析龈沟液miR-155、miR-223表达水平与牙周临床指标、口腔龈下菌群、外周血Th17/Treg以及血清IL-17、TGF-β的相关性。结果：慢性牙周炎伴T2DM组、单纯慢性牙周炎组龈沟液miR-155、miR-223表达水平高于对照组（P＜0.05）,且慢性牙周炎伴T2DM组高于单纯慢性牙周炎组（P＜0.05）。慢性牙周炎伴T2DM组龈沟出血指数（SBI）、菌斑指数（PLI）、探诊深度（PD）、附着丧失（AL）、牙龈卟啉单胞菌、二氧化碳噬纤维菌、中间普氏菌、变黑普氏菌数量、外周血Th17细胞占比、Th17/Treg比值、血清IL-17水平高于单纯慢性牙周炎组（P＜0.05）,外周血Treg细胞占比低于单纯慢性牙周炎组（P＜0.05）。龈沟液miR-155、miR-223表达水平与PLI、SBI、AL、PD、牙龈卟啉单胞菌、二氧化碳噬纤维菌、中间普氏菌、变黑普氏菌数量、外周血Th17细胞占比、Th17/Treg比值、血清IL-17水平呈正相关（P＜0.05）,与外周血Treg细胞占比呈负相关（P＜0.05）。结论：慢性牙周炎伴T2DM患者龈沟液中miR-155、miR-233表达均上调,且与牙周组织破坏程度、龈下菌群紊乱和Th17/Treg失衡有关。     

慢性牙周炎患者血清CGRP、PGE2、CCL20与牙周临床指标和Th17/Treg失衡的相关性分析

摘要 目的：探究慢性牙周炎患者血清降钙素基因相关肽（CGRP）、前列腺素E₂（PGE₂）、CC趋化因子配体20（CCL20）与牙周临床指标和辅助性T淋巴细胞17/调节性T淋巴细胞（Th17/Treg）失衡的相关性。方法：选取2020年5月-2022年5月海南省妇女儿童医学中心收治的91例慢性牙周炎患者,根据其严重程度分为轻度组（39例）、中度组（36例）、重度组（16例）,比较三组血清CGRP、PGE₂、CCL20、牙周临床指标[出血指数（BI）、探诊深度（PD）、附着丧失（AL）、菌斑指数（PLI）]、外周血Th17细胞比例、Treg细胞比例、Th17/Treg比值,采用Pearson相关分析血清CGRP、PGE₂、CCL20与牙周临床指标和Th17/Treg失衡的相关性。结果：与轻度组比较,中度组、重度组血清CGRP、Treg细胞比例显著降低（P＜0.05）,血清PGE₂、CCL20、BI、PD、PLI、AL、Th17细胞比例、Th17/Treg比值显著增高（P＜0.05）;与中度组比较,重度组血清CGRP、Treg细胞比例显著降低（P＜0.05）,血清PGE₂、CCL20、BI、PD、PLI、AL、Th17细胞比例、Th17/Treg比值显著增高（P＜0.05）。相关性结果提示,血清PGE₂、CCL20水平与BI、PD、PLI、AL、Th17细胞比例、Th17/Treg比值呈正相关（P＜0.05）,与Treg细胞比例呈负相关（P＜0.05）;血清CGRP水平与BI、PD、PLI、AL、Th17细胞比例、Th17/Treg比值呈负相关（P＜0.05）,与Treg细胞比例呈正相关（P＜0.05）。结论：慢性牙周炎患者血清CGRP、PGE₂、CCL20水平与疾病严重程度、牙周临床指标及Th17/Treg失衡显著相关,血清CGRP、PGE₂、CCL20可能通过影响Th17/Treg平衡参与慢性牙周炎的发生和发展。     

侵袭性牙周炎伴错牙合畸形患者牙周-正畸联合治疗前后血清SAA、leptin的变化及与牙周指标和辅助性T细胞亚群的相关性分析

摘要 目的：探讨侵袭性牙周炎伴错牙合畸形患者牙周-正畸联合治疗前后血清淀粉样蛋白A（SAA）、瘦素（leptin）的变化及与牙周指标和辅助性T细胞（Th）亚群的相关性。方法：选择2020年6月-2022年8月解放军总医院京中医疗区黄寺门诊部口腔科收治的80例侵袭性牙周炎伴错牙合畸形患者（牙周炎组）和65例于口腔门诊检查的健康志愿者（对照组）。所有患者均接受牙周-正畸联合治疗,治疗前后分别检测血清SAA、leptin水平以及外周血中Th1、Th2、Th17细胞占比,并评估牙周指标变化。Pearson相关性分析血清SAA、leptin水平与牙周指标以及外周血中Th1、Th2、Th17细胞占比的相关性。结果：牙周炎组治疗前血清SAA、leptin水平,外周血Th1、Th17细胞占比,出血指数（SBI）、菌斑指数（PLI）、附着丧失（AL）、牙周探诊深度（PD）高于对照组（P＜0.05）,外周血Th2细胞占比低于对照组（P＜0.05）。牙周炎组治疗后血清SAA、leptin水平,外周血Th1、Th17细胞占比,PLI、SBI、AL、PD较治疗前降低（P＜0.05）,外周血Th2细胞占比较治疗前增高（P＜0.05）。牙周炎组血清SAA、leptin与PLI、SBI、AL、PD,外周血Th1、Th17细胞占比呈正相关,与外周血Th2细胞占比呈负相关（P＜0.05）。结论：侵袭性牙周炎伴错牙合畸形患者血清SAA、leptin水平增高,经牙周-正畸联合治疗后下降,高水平SAA、leptin与牙周组织破坏程度以及Th亚群紊乱有关,检测血清SAA、leptin水平可评估侵袭性牙周炎牙周组织破坏程度以及细胞免疫状态。     

慢性牙周炎合并2型糖尿病患者龈沟液miR-21、miR-34a表达水平与牙周指标和Th1/Th2/Th17失衡的关系研究

摘要 目的：探讨慢性牙周炎（CP）合并2型糖尿病（T2DM）患者龈沟液微小核糖核酸（miR）-21、miR-34a表达水平与牙周指标和辅助性T细胞（Th）1/Th2/Th17失衡的关系。方法：选取2020年3月～2022年3月首都医科大学附属北京世纪坛医院口腔科收治的114例CP患者，根据是否合并T2DM分为CP合并T2DM组36例和单纯CP组78例，另选取60名健康体检者为对照组。对比三组牙周指标、龈沟液miR-21、miR-34a表达和外周血Th1、Th2、Th17、Th1/Th2/Th17、血清Th1、Th2、Th17相关细胞因子水平，采用Spearman相关性分析CP合并T2DM患者龈沟液miR-21、miR-34a表达与牙周指标和外周血Th1、Th2、Th17、Th1/Th2/Th17及其相关细胞因子水平的相关性。采用Pearson/Spearman相关性分析CP合并T2DM患者牙周指标与外周血Th1、Th2、Th17、Th1/Th2/Th17及其相关细胞因子水平的相关性。结果：对照组、单纯CP组、CP合并T2DM组菌斑指数（PLI）、牙龈出血指数（BI）、附着丧失（AL）、探诊深度（PD）依次增加，龈沟液miR-21和外周血Th2及血清白细胞介素（IL）-2、干扰素-γ（INF-γ）依次降低，龈沟液miR-34a和外周血Th1、Th17、Th1/Th2/Th17及血清IL-4、IL-10、IL-17、肿瘤坏死因子-α（TNF-α）依次升高（P＜0.05）。Spearman相关性分析显示，CP合并T2DM患者龈沟液miR-21表达与PLI、BI、AL、PD和外周血Th1、Th17、Th1/Th2/Th17及血清IL-4、IL-10、IL-17、TNF-α呈负相关，与外周血Th2和血清IL-2、INF-γ呈正相关（P＜0.05）；而miR-34a则与之相反。Pearson/Spearman相关性分析显示，CP合并T2DM患者PLI、BI、AL、PD与外周血Th1、Th17、Th1/Th2/Th17和血清IL-4、IL-10、IL-17、TNF-α呈正相关，与外周血Th2和血清IL-2、INF-γ呈负相关（P＜0.05）。结论：CP合并T2DM患者龈沟液miR-21低表达和miR-34a高表达，与牙周状况差有关，可能通过调节Th1/Th2/Th17失衡参与CP合并T2DM进展。     

支气管哮喘患儿血清半乳糖凝集素3、类胰蛋白酶、25-羟维生素D3与肺功能和生活质量的相关性分析

摘要 目的：探讨支气管哮喘（简称"哮喘"）患儿血清半乳糖凝集素3（Gal-3）、类胰蛋白酶、25-羟维生素D₃ [25（OH）D₃]与肺功能和生活质量的相关性。方法：选取我院2018年2月～2021年3月收治的136例哮喘患儿为研究组，行肺功能检测，将其按照病情严重程度分作轻度组63例、中度组41例及重度组32例。另取同期健康体检儿童40例作为对照组。检测并比较各组血清Gal-3、类胰蛋白酶、25（OH）D₃水平。对比各组哮喘患儿肺功能指标，以中文版儿科哮喘生命质量调查问卷(PAQLQ)对哮喘患儿进行生活质量评估。并以Pearson相关性分析哮喘患儿血清Gal-3、类胰蛋白酶、25（OH）D₃水平与肺功能和生活质量的关系。结果：研究组的血清Gal-3、类胰蛋白酶水平均高于对照组，且轻度组、中度组、重度组逐渐升高；血清25（OH）D₃水平均低于对照组，轻度组、中度组、重度组逐渐下降（P＜0.05）。中度组及重度组第1秒用力呼气容积占预计值百分比（FEV₁%pred）、呼吸峰流速占预计值百分比（PEF%pred）、第1秒用力呼气容积/用力肺活量（FEV₁/FVC）以及PAQLQ各项评分均低于轻度组，且重度组低于中度组，差异均有统计学意义（P＜0.05）。经Pearson相关性分析可得：哮喘患儿血清Gal-3、类胰蛋白酶水平与各项肺功能指标以及PAQLQ各项评分均呈负相关，而血清25（OH）D₃水平与与各项肺功能指标以及PAQLQ各项评分均呈正相关（均P＜0.05）。结论：血清Gal-3、类胰蛋白酶、25（OH）D₃水平与哮喘患儿的肺功能以及生活质量密切相关，可能成为评估其病情及生活质量的可靠指标。     

慢性牙周炎合并2型糖尿病患者龈沟液Sirtuin-1、Sirtuin-6的变化及临床价值研究

摘要 目的：探讨慢性牙周炎（CP）合并2型糖尿病（T2DM）患者龈沟液沉默信息调节因子-1（Sirtuin-1）、Sirtuin-6的变化和临床价值。方法：选择2020年3月至2023年3月中国人民解放军联勤保障部队第九七〇医院收治的147例CP合并T2DM患者（T2DM组）,128例单纯CP患者（CP组）和121例健康体检者（对照组）。根据牙周检查结果将T2DM组患者分为轻度组（n=49）、中度组（n=67）、重度组（n=31）。检测受试者龈沟液中Sirtuin-1、Sirtuin-6水平以及外周血单核细胞核苷酸结合寡聚化结构域样受体热蛋白结构域亚家族成员3（NLRP3）信使核糖核酸（mRNA）、程序性细胞死亡相关斑点样蛋白（ASC）mRNA、半胱氨酸蛋白酶1（Caspase-1）mRNA表达,并评估牙周临床指标。Pearson分析CP合并T2DM患者龈沟液Sirtuin-1、Sirtuin-6水平与牙周临床指标、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达的相关性。受试者工作特征（ROC）曲线分析龈沟液Sirtuin-1、Sirtuin-6诊断CP合并T2DM的价值。结果：T2DM组龈沟液Sirtuin-1、Sirtuin-6水平低于CP组和对照组（P＜0.05）,出血指数（SBI）、牙周袋探诊深度（PD）、牙龈指数（GI）、菌斑指数（PLI）、附着丧失（AL）、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于CP组和对照组（P＜0.05）。CP组龈沟液Sirtuin-1、Sirtuin-6水平低于和对照组（P＜0.05）,GI、SBI、PLI、PD、AL、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于对照组（P＜0.05）。重度组龈沟液Sirtuin-1、Sirtuin-6水平低于中度组和轻度组（P＜0.05）,GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于中度组和轻度组（P＜0.05）。中度组龈沟液Sirtuin-1、Sirtuin-6水平低于轻度组（P＜0.05）,GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达高于轻度组（P＜0.05）。CP合并T2DM患者龈沟液Sirtuin-1、 Sirtuin-6水平与GI、PLI、SBI、AL、PD、外周血单核细胞NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达均呈负相关（P＜0.05）。龈沟液Sirtuin-1、 Sirtuin-6诊断CP合并T2DM的曲线下面积（AUC）为0.787、0.806,联合诊断AUC为0.912,高于单独诊断。结论：CP合并T2DM患者龈沟液中Sirtuin-1、Sirtuin-6水平降低,且与牙周组织破坏程度加重、NLRP3炎症小体激活有关。龈沟液Sirtuin-1联合Sirtuin-6在CP合并T2DM诊断中具有较高价值。     

慢性牙周炎伴咬合创伤患者龈沟液Sclerostin、SLIT3与牙周临床指标和RANKL-OPG系统的相关性研究

摘要 目的：探讨慢性牙周炎伴咬合创伤患者龈沟液骨硬化蛋白（Sclerostin）、神经轴突导向因子3（SLIT3）与牙周临床指标和核因子-κb受体活化因子配体（RANKL）-骨保护蛋白（OPG）系统的相关性。方法：选择2021年1月至2023年1月我院收治的123例慢性牙周炎伴咬合创伤患者（创伤组）和101例不伴咬合创伤的慢性牙周炎患者（对照组）。治疗前,检测龈沟液中Sclerostin、SLIT3、RANKL、OPG水平,计算RANKL/OPG比值,评估临床牙周指标出血指数（SBI）、菌斑指数（PLI）、探诊深度（PD）、附着丧失（AL）。分析Sclerostin、SLIT3与牙周临床指标、RANKL、OPG、RANKL/OPG之间的相关性。结果：创伤组治疗前龈沟液中Sclerostin、SLIT3、RANKL水平,RANKL/OPG,PLI、SBI、AL、PD高于对照组（P＜0.05）,OPG低于对照组（P＜0.05）。创伤组治疗前龈沟液中Sclerostin、SLIT3与PLI、SBI、AL、PD、RANKL、RANKL/OPG呈正相关（P＜0.05）,与OPG呈负相关（P＜0.05）。结论：慢性牙周炎伴咬合创伤患者龈沟液中Sclerostin、SLIT3水平显著增高,且与牙周临床指标异常、RANKL/OPG增加有关。     

牙龈卟啉单胞菌感染对慢性牙周炎患者牙周指标、NLRP-3炎性小体通路和Th17/Treg细胞平衡的影响

摘要 目的：探讨牙龈卟啉单胞菌（PG）感染对慢性牙周炎（CP）患者牙周指标、Nod样受体蛋白-3（NLRP-3）炎性小体通路和辅助性T细胞17（Th17）/调节性T细胞（Treg）平衡的影响。方法：选择2018年6月至2021年11月在本院进行拔牙治疗的慢性牙周炎患者106例为CP组,根据是否感染PG分为感染组和未感染组;另选择因阻生齿、错位牙在本院进行治疗的牙周健康患者63例为对照组。记录所有对象的探诊深度（PD）、菌斑指数（PLI）、出血指数（BI）、附着丧失（AL）,采用聚合酶链式反应（PCR）技术检测PG感染率,采用RT-PCR技术检测牙周膜组织中NLRP-3 mRNA,接头蛋白凋亡相关斑点样蛋白（ASC） mRNA、半胱氨酸天冬氨酸酶-1（Caspase-1） mRNA的表达量,采用FACSCalibur流式细胞仪检测外周血Th17、Terg 水平并计算Th17/Treg,比较两组各检测指标水平。结果：对照组PG感染阳性率为17.46%,低于CP组的79.25%,两组比较差异有统计学意义（P＜0.05）。感染组PD、PLI、BI、AL水平均高于非感染组,差异有统计学意义（P＜0.05）。感染组NLRP-3 mRNA、ASC mRNA、Caspase-1 mRNA表达量均高于非感染组,差异有统计学意义（P＜0.05）。感染组外周血Th17细胞、Th17/Treg水平高于非感染组,Treg水平低于非感染组,差异有统计学意义（P＜0.05）。结论：CP患者多数存在PG感染,PG感染对患者的牙周健康、炎症及免疫有明显的影响。     

Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively

We have separated and quantified two new minor hemoglobins named HbA_1d3a and HbA_1d3b. The level of HbA_1d3a was significantly higher in uremic than in non-uremic patients (3.00 ± 0.50% vs. 1.28 ± 0.26% of total hemoglobin). It correlated well with carbamylated hemoglobin (r=0.80, n=81, p<0.002) and with plasma urea concentration (r=0.78, n=81, p<0.002). These data and the electrospray ionization mass spectrometric analysis provide strong evidence that HbA_1d3a is an α-chain modified by carbamylation. The HbA_1d3b level in the diabetic patients was found to be 1.6-fold that in non-diabetic subjects (3.00 ± 0.49 vs. 1.90 ± 0.33). This was attributed to HbA_1d3 modified by glycation. Indeed HbA_1d3b correlated significantly with HbA_1c (r=0.71, p<0.002) and with serum glucose level (r=0.62, p<0.002). These two new minor hemoglobins may serve as complements for the objective assessment of averagd long-term uremia and glycemia in uremic and diabetic patients.     

温带针阔混交林土壤碳氮气体通量的主控因子与耦合关系

中高纬度森林地区由于气候条件变化剧烈,土壤温室气体排放量的估算存在很大的不确定性,并且不同碳氮气体通量的主控因子与耦合关系尚不明确。以长白山温带针阔混交林为研究对象,采用静态箱-气相色谱法连续4a(2005—2009年)测定土壤二氧化碳(CO2)、甲烷(CH4)和氧化亚氮(N2O)净交换通量以及温度、水分等相关环境因子。研究结果表明:温带针阔混交林土壤整体上表现为CO2和N2O的排放源和CH4的吸收汇。土壤CH4、CO2和N2O通量的年均值分别为-1.3 kg CH4hm-2a-1、15102.2 kg CO2hm-2a-1和6.13 kg N2O hm-2a-1。土壤CO2通量呈现明显的季节性规律,主要受土壤温度的影响,水分次之;土壤CH4通量的季节变化不明显,与土壤水分显著正相关;土壤N2O通量季节变化与土壤CO2通量相似,与土壤水分、温度显著正相关。土壤CO2通量和CH4通量不存在任何类型的耦合关系,与N2O通量也不存在耦合关系;土壤CH4和N2O通量之间表现为消长型耦合关系。这项研究显示温带针阔混交林土壤碳氮气体通量主要受环境因子驱动,不同气体通量产生与消耗之间存在复杂的耦合关系,下一步研究需要深入探讨环境变化对其耦合关系的影响以及内在的生物驱动机制。     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Vitamin D₃ (VD₃) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD₃ into its physiologically active forms, namely, 25(OH)VD₃ or 1α,25(OH)₂VD₃. In this study, we isolated and characterized Vdh (vitamin D₃ hydroxylase), which hydroxylates VD₃ from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V_max values for VD₃ 25-hydroxylation and 25(OH)VD₃ 1α-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD₃. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD₃ 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD₃ into 25(OH)VD₃ was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD₃ by a single cytochrome P450.     

CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells

We have shown earlier a requirement for Ca²⁺ and calmodulin (CaM) in the H₂O₂-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H₂O₂-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKIIα. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H₂O₂-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H₂O₂, calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281–309) of CaMKII and with siRNA of CaMKIIα attenuated the H₂O₂-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H₂O₂-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKIIα siRNA abolished the H₂O₂-induced IGF-1R phosphorylation. H₂O₂ treatment also induced Thr²⁸⁶ phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H₂O₂ on ERK1/2, PKB, and IGF-1R phosphorylation.     

Ammonia-induced Na,K-ATPase/ouabain-mediated EGF receptor transactivation,MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling

Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3 mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α₂ isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present cell culture study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK_1/2 and PI3K/AKT; interaction between EGF receptor, α₁, and α₂ isoforms of Na,K-ATPase, Src, ERK_1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that (i) ammonia increases EGF receptor phosphorylation at EGFR⁸⁴⁵ and EGFR¹⁰⁶⁸; (ii) ammonia-induced ERK_1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; (iii) AKT phosphorylation occurs upstream of ERK_1/2 phosphorylation; (iv) ammonia stimulates association between the α₁ Na,K-ATPase isoform, Src, EGF receptor, ERK_1/2, AKT and caveolin-1; (v) ammonia-induced ROS production might occur later than EGFR transactivation; (vi) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and (vii) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK_1/2, but in response to 3 mM ammonia it does not appear until after 12 h. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at this concentration reflects required ouabain-induced oxidative damage of the ion and water cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic brain disorders.     

Utilization of Serine, Leucine, Isoleucine, and Valine byThermoanaerobacter brockiiin the Presence of Thiosulfate orMethanobacteriumsp. as Electron Acceptors

Thermoanaerobacter brockiifermented serine to acetate and ethanol. It oxidized leucine to isovalerate, isoleucine to 2-methylbutyrate, and valine to isobutyrate only in the presence of thiosulfate, or when co-cultured withMethanobacteriumsp. This oxidative deamination was rendered thermodynamically possible by the ability ofT. brockiito reduce thiosulfate to sulfide or the transfer of reducing equivalents to the hydrogenotrophic methanogen. The results suggest thatT. brockiimay be of ecological significance in thermal environments in the turnover of amino acids, especially with thiosulfate or H₂-utilizing methanogens are present.     

A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: Purification,proteomic, functional and structural characterizations

A phospholipase A₂ was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA₂. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA₂ was a new snake venom acidic PLA₂. Seven peptides were sequenced from Akbu-PLA₂ by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA₂ shared homolog peptides of phospholipases A₂ from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA₂ has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA₂ showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA₂ was shown to contain one Ca²⁺ per monomer by ICP-AES measurement. The Ca²⁺ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA₂. Ca²⁺ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA₂. The hydrolytic activity of Akbu-PLA₂ was accelerated due to the addition of Ca²⁺ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA₂ was found to be difficult to eliminate for the purification of Akbu-PLA₂. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA₂, which indicated that it should be a homodimer of Akbu-PLA₂. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA₂ monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA₂ monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca²⁺ ion was found to be able to promote the dimer formation of Akbu-PLA₂. We conclude that a novel PLA₂ was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA₂ reveal new threads and provide valuable inputs for the study of snake venom phospholipases A₂.     

间隙连接及其调控因素

概述了间隙连接的结构和功能,以及pH值、电压、生长因子对它的调节作用.间隙连接作为相邻细胞间信息物质通道,起着传递细胞信息,协调细胞群体功能的作用,但是其形成过程以及对机体生理功能影响的研究有待进一步深入.pH值降低可引起间隙连接通道的关闭,电压升高降低通道的导电性,生长因子可通过影响间隙连接蛋白的形成和降解、促使间隙连接蛋白磷酸化调节其通透性.     

Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.