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。     

龈沟液Chemerin、APN、25(OH)D3与伴2型糖尿病的慢性牙周炎患者牙周指标和Th17/Treg失衡的关系

摘要 目的：探讨龈沟液趋化素（Chemerin）、脂联素（APN）、25-羟维生素D3 [25(OH)D3]与伴2型糖尿病（T2DM）的慢性牙周炎（CP）患者牙周指标和Th17/Treg失衡的关系。方法：选择2021年1月至2022年1月我院口腔科门诊接诊的125例伴T2DM的CP患者,根据CP病情严重程度分为轻度组（42例）、中度组（53例）和重度组（30例）。检测龈沟液中Chemerin、APN、25(OH)D₃水平以及外周血中Th17细胞占比、Treg细胞占比,计算Th17/Treg比值,记录出血指数（SBI）、菌斑指数（PLI）、附着丧失（AL）、牙周袋探诊深度（PD）。分析龈沟液中Chemerin、APN、25(OH)D₃与牙周指标、外周血Th17/Treg的相关性。结果：重度组龈沟液 Chemerin水平和外周血中Th17细胞占比、Th17/Treg比值、PLI、SBI、AL、PD高于中度组和轻度组（P＜0.05）,龈沟液APN、25(OH)D₃水平,外周血Treg细胞占比低于中度组和轻度组（P＜0.05）。龈沟液 Chemerin与PLI、SBI、AL、PD、外周血Th17细胞占比、Th17/Treg比值呈正相关（P＜0.05）,与Treg细胞占比呈负相关（P＜0.05）;龈沟液APN、25(OH)D₃与PLI、SBI、AL、PD、外周血中Th17细胞占比、Th17/Treg比值呈负相关（P＜0.05）,与Treg细胞占比呈正相关（P＜0.05）。结论：伴T2DM 的CP患者龈沟液中Chemerin水平增高,APN、25(OH)D₃水平降低,且与牙周指标和Th17/Treg失衡有关。     

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。     

吸烟对种植体周围炎患者龈下菌群、龈沟液炎症因子和RANKL/OPG比值的影响

摘要 目的：探讨吸烟对种植体周围炎患者龈下菌群分布、龈沟液炎症因子白细胞介素-4（IL-4）、白细胞介素-5（IL-5）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、白细胞介素-17（IL-17）和核因子-κB受体活化因子配体/骨保护素（RANKL/OPG）比值的影响。方法：选择2019年3月至2022年3月首都医科大学附属北京友谊医院口腔科收治的种植体周围炎患者99例（共151颗种植体）,根据是否吸烟分为吸烟组（45例,68颗种植体）和非吸烟组（54例,83颗种植体）,比较两组患者种植体牙周改良菌斑指数（mPLI）、牙龈出血指数（GBI）、种植体周围探诊深度（PPD）,采集两组龈下菌斑进行细胞培养并进行菌种鉴定,分析两组龈下菌群分布情况,比较两组龈沟液中IL-4、IL-5、IL-6、IL-8、IL-17、RANKL/OPG比值。结果：吸烟组mPLI、GBI、PPD高于不吸烟组（P＜0.05）。吸烟组厌氧菌检出率高于非吸烟组（P＜0.05）,有益菌检出率低于非吸烟组（P＜0.05）,两组需氧菌检出率比较差异无统计学意义（P＞0.05）。吸烟组龈沟液IL-4、IL-5、IL-6、IL-8、IL-17水平均高于非吸烟组（P＜0.05）。吸烟组龈沟液RANKL水平、RANKL/OPG比值高于非吸烟组（P＜0.05）,OPG水平低于非吸烟组（P＜0.05）。结论：吸烟可导致种植体周围炎患者龈下厌氧菌增加,加重炎症反应,增加牙槽骨吸收风险。     

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。     

羌活地黄汤对佐剂性类风湿关节炎大鼠作用机制研究

摘要 目的：研究羌活地黄汤对佐剂性类风湿关节炎（RA）大鼠辅助性T细胞/调节性T细胞（Th17/Treg）失衡、血清基质金属蛋白酶-3（MMP-3）、基质金属蛋白酶-13（MMP-13）以及滑膜组织血管内皮生长因子（VEGF）、核因子-κB受体活化因子配基（RANKL） mRNA表达的影响。方法：取40只SD级大鼠进行研究，将其以随机数字表法分为正常对照组、模型组、羌活地黄汤组以及甲氨蝶呤组，每组各10只。除正常对照组外，其余各组大鼠均建立佐剂性RA动物模型。羌活地黄汤组予以羌活地黄汤灌胃干预，甲氨蝶呤组予以甲氨蝶呤灌胃干预，正常对照组及模型组均予以等量生理盐水灌胃干预。各组均进行为期28d的干预，比较各组体重、关节炎指数及关节肿胀度、外周血Th17/Treg相关指标、血清MMP-3、MMP-13水平、滑膜组织VEGF、RANKL mRNA表达。结果：模型组体重低于正常对照组，羌活地黄汤组、甲氨蝶呤组关节炎指数及关节肿胀度均低于模型组，而体重高于模型组（均P＜0.05）。模型组、羌活地黄汤组、甲氨蝶呤组的外周血CD4+IL-17+T、Th17/Treg均高于正常对照组，而Foxp3+CD4+CD25+Treg低于正常对照组，且羌活地黄汤组、甲氨蝶呤组的外周血CD4+IL-17+T、Th17/Treg均低于模型组，而Foxp3+CD4+CD25+Treg高于模型组（均P＜0.05）。模型组、羌活地黄汤组、甲氨蝶呤组的血清MMP-3、MMP-13水平均高于正常对照组，而羌活地黄汤组、甲氨蝶呤组的血清MMP-3、MMP-13水平均低于模型组（均P＜0.05）。模型组、羌活地黄汤组、甲氨蝶呤组的滑膜组织VEGF、RANKL mRNA表达水平均高于正常对照组，而羌活地黄汤组、甲氨蝶呤组的滑膜组织VEGF、RANKL mRNA表达水平均低于模型组（均P＜0.05）。结论：羌活地黄汤可改善佐剂性RA大鼠的症状以及Th17/Treg失衡状态，且有效改善其血清MMP-3、MMP-13水平以及滑膜组织VEGF、RANKL mRNA表达。     

龈沟液Melatonin、PTX3与老年牙隐裂伴慢性牙髓炎患者炎症因子和治疗效果的关系研究

摘要 目的：探究龈沟液褪黑素（Melatonin）、正五聚体蛋白3（PTX3）与老年牙隐裂伴慢性牙髓炎（CP）患者炎症因子和治疗效果的关系。方法：选择2021年6月-2022年6月我院收治的165例老年牙隐裂伴CP患者纳入研究组,选取同期在我院实施正畸牙拔除的健康受试者100例纳入对照组。比较两组龈沟液Melatonin、PTX3及炎症因子[肿瘤坏死因子-α （TNF-α）、白细胞介素（IL-6）、C反应蛋白（CRP）]水平。Pearson检验分析老年牙隐裂伴CP患者龈沟液Melatonin、PTX3水平与炎症因子的相关性。165例老年牙隐裂伴CP患者接受根管治疗+全冠修复后随访1年,根据治疗效果分为有效组和无效组,比较两组临床资料。多因素Logistic回归模型分析老年牙隐裂伴CP患者治疗无效的影响因素;受试者工作特征（ROC）曲线分析龈沟液Melatonin、PTX3对老年牙隐裂伴CP患者治疗无效的预测价值。结果：与对照组比较,研究组龈沟液中Melatonin水平降低（P＜0.05）,PTX3、TNF-α、CRP、IL-6水平升高（P＜0.05）。Pearson检验显示,老年牙隐裂伴CP患者龈沟液PTX3与TNF-α、CRP、IL-6呈正相关（P＜0.05）,龈沟液Melatonin与TNF-α、CRP、IL-6呈负相关（P＜0.05）。165例老年牙隐裂伴CP患者,根管治疗+全冠修复治疗结束后随访1年,失访3例,最终共162例患者完成随访。162例患者中成功122例、好转26例、失败14例,无效率为8.64%（14/162）。与有效组比较,无效组裂纹达髓底占比、探诊深度＞5 mm占比、PTX3水平显著升高（P＜0.05）,Melatonin水平显著降低（P＜0.05）。多因素Logistic回归模型结果显示,裂纹深度达髓底、探诊深度＞5 mm、PTX3水平升高是老年牙隐裂伴CP患者治疗无效的危险因素（P＜0.05）,Melatonin水平升高为其保护因素（P＜0.05）。ROC曲线显示,龈沟液Melatonin联合PTX3检测预测老年牙隐裂伴CP患者治疗无效的曲线下面积（AUC）、灵敏度、特异度为0.803、0.857、0.777。结论：龈沟液Melatonin水平降低、PTX3水平升高与老年牙隐裂伴CP患者炎症反应有关,且Melatonin水平越低、PTX3水平越高者治疗效果越差,Melatonin联合PTX3检测对于根管治疗+全冠修复治疗效果有较高的预测价值。     

龈沟液CRP、IL-17、RANKL与种植体周围炎患者牙周临床指标的相关性分析及其联合检测对治疗后预后的评估价值

摘要 目的：分析龈沟液C反应蛋白（CRP）、白细胞介素-17（IL-17）及核因子-kB受体活化因子配体（RANKL）与种植体周围炎（PI）患者牙周临床指标的相关性及探讨其联合检测对治疗后预后的评估价值。方法：选取2018年1月～2022年1月我院收治的80例PI患者,记作炎症组。另取同期种植体健康患者80例作为健康组。检测并比较两组龈沟液CRP、IL-17及RANKL水平,牙周临床指标。并以Pearson相关性分析龈沟液CRP、IL-17及RANKL与牙周临床指标的相关性。此外,将所有PI患者按照急性生理学与慢性健康状况评分Ⅱ（APACHE Ⅱ）评分的差异分为预后良好组27例与预后不良组53例,比较两组龈沟液CRP、IL-17及RANKL水平。通过受试者工作特征（ROC）曲线分析上述三项指标联合预测PI患者治疗后预后不良的效能。结果：炎症组龈沟液CRP、IL-17及RANKL水平均高于健康组（均P＜0.05）。炎症组改良牙龈指数（mGI）、改良龈沟出血指数（mSBI）评分以及探诊深度（PD）均高于健康组（均P＜0.05）。经Pearson相关性分析发现,龈沟液CRP、IL-17及RANKL水平与mGI、mSBI评分及PD均呈正相关关系（均P＜0.05）。预后不良组龈沟液CRP、IL-17及RANKL水平均高于预后良好组（均P＜0.05）。经ROC曲线分析发现,龈沟液CRP、IL-17及RANKL联合预测PI患者治疗后预后不良的曲线下面积（0.95CI）为0.851（0.752～0.944）,优于上述三项指标单独预测。结论：龈沟液CRP、IL-17及RANKL与PI患者mGI、mSBI评分以及PD均密切相关,检测三指标有助于评估患者病情及治疗后预后。     

Chiral separation and quantification of R/S-amphetamine, R/S-methamphetamine, R/S-MDA, R/S-MDMA, and R/S-MDEA in whole blood by GC-EI-MS

The enantioselective composition of the amphetamines is of interest, as the enantiomers show differences in their pharmacological effects and several methods for chiral separation of amphetamines have been described. Only a few methods have used whole blood as matrix and none of these separates both classic amphetamines (amphetamine and methamphetamine) and designer amphetamines (MDA, MDMA and MDEA). The aim of this study was, therefore, to develop a method for enantioselective analysis of AM, MA, MDA, MDMA, and MDEA in whole blood. The amphetamines were extracted from 0.5 g of whole blood by liquid-liquid extraction. After derivatization with R-MTPCl, the resulting diastereomers were separated by GC on a HP-5MS column and detected by SIM-MS. R-MTPCl was used as derivatization reagent because of the stability of this reagent and good separation of these analytes. Through the method, development time and temperature of the derivatization were optimized, and by admixture of 0.02% triethylamine it became possible to detect the amphetamines in adequately low concentrations as more analytes were derivatized. The method was validated and it was linear from 0.004 to 3 microg/g per enantiomer. The accuracy was within 91-115%, while the repeatability and reproducibility were < or =15% R.S.D. A method suitable for enantioselective separation and analysis of the amphetamines has been achieved, and the method was applied to analysis of whole blood samples originating from traffic and criminal cases and post mortem cases.     

Synthesis, characterization, cytotoxicity and antibacterial studies of chitosan, O-carboxymethyl and N,O-carboxymethyl chitosan nanoparticles

Chitosan (CS) is a naturally occurring biopolymer. It has important biological properties such as biocompatibility, antifungal and antibacterial activity, wound healing ability, anticancerous property, anticholesteremic properties, and immunoenhancing effect. Recently, CS nanoparticles have been used for biomedical applications. However, due to the limited solubility of CS in water its water-soluble derivatives are preferred for the above said applications. In this work, the nanoparticles of CS and its water-soluble derivatives such as O-carboxymethyl chitosan (O-CMC) and N,O-carboxymethyl chitosan (N,O-CMC) was synthesized and characterized. In addition, cytotoxicity and antibacterial activity of the prepared nanoparticles was also evaluated for biomedical applications.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.␣coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene. Received: 15 April 1998 / Accepted: 3 August 1998     

Anthocyanins of Cephaelis, Cynomorium, Euterpe, Lavatera and Pinanga

The two malonylated pigments, malonylmalvin and malvidin 3-malonylglucoside, were identified in petals of Lavatera maritima, which belongs to the Malvaceae, a family known to synthesise such pigments. Zwitterionic anthocyanins could not be detected in four other newly examined sources and common unacylated pigments were recorded. Thus, the fruits of the palms Euterpe edulis and Pinanga polymorpha have a mixture of cyanidin 3-glucoside and cyanidin 3-rutinoside, while the fruit of Cephaelis subcoriacea is coloured by cyanidin 3-glucoside. The latter pigment was also obtained from the reddish brown inflorescence of the parasitic plant Cynomorium coccineum.     

Synthesis, characterization and redox behaviour of benzoyldiazenido- and oxorhenium complexes bearing N,N- and S,S-type ligands

The reactions of [ReCl₂{η²-N₂C(O)Ph}(PPh₃)₂] (1) with 2-aminopyrimidine (H₂Npyrm), 2,2′-bipyridine (bpy) and tetraethylthiuram disulfide (tds), in MeOH upon reflux, lead to the new η¹-(benzoyldiazenido)rhenium(III) complexes [ReCl{η¹-N₂C(O)Ph}(HNpyrm)(PPh₃)₂] (2) and [ReCl₂{η¹-N₂C(O)Ph}(bpy)(PPh₃)] (3), and the known oxo(diethyldithiocarbamato)dirhenium(v) complex [Re₂O₂(μ-O){Et₂NC(S)S}₄] (4), respectively. The Et₂NC(S)S ligands in 4 result from S-S bond rupture of tds molecules. The obtained compounds have been characterized by IR, ¹H, ³¹P{¹H} and ¹³C{¹H} NMR spectroscopies, FAB⁺-MS, elemental and single-crystal X-ray diffraction (for 2 and 4) analyses. Complex 2 represents the first structurally characterized Re compound derived from 2-aminopyrimidine. Besides, the redox behaviour of 2-4 in CH₂Cl₂ solution has been studied by cyclic voltammetry, and the Lever electrochemical ligand parameter (E_L) has been estimated, for the first time, for HNpyrm. The electrochemical results are discussed in terms of electronic properties of the Re centres and the ligands.     

Preparation, properties and crystal structure of two isomers of R,R,S,S- and R,S,R,S-[chloro(1,3,10,12,16,19-hexaazatetracyclo[17,3,1,1,0]tetracosane)copper(II)]

Two isomers (R,S,R,S- and R,R,S,S-) of five coordinate complex [Cu(L)Cl]⁺ have been separated and characterised. These two isomers have significantly different spectrochemical and electrochemical properties. Absorption maximum of R,S,R,S-[Cu(L)Cl]⁺ shifts to longer wavelength and its reduction potential shifts to more positive direction comparing those of R,R,S,S-[Cu(L)Cl]⁺. R,S,R,S-[Cu(L)Cl]⁺ is significantly distorted to trigonal-bipyramidal structure, whereas R,R,S,S-[Cu(L)Cl]⁺ retains almost square-planar geometry. The average bond distance of Cu-N in basal plane of R,S,R,S-[Cu(L)Cl]⁺ is longer by 0.024 Å than that of R,R,S,S-[Cu(L)Cl]⁺, whereas the bond distance of Cu-Cl in former is shorter by 0.200 Å than that in latter. The isolated square-planar complexes of R,R,S,S- and R,S,R,S-[Cu(L)](ClO₄)₂ are converted to the R,R,S,S- and R,S,R,S-[Cu(L)Cl]⁺ by the addition of Cl⁻ in nitromethane solution with the rate constants, k=1.70 (±0.02) and 8.31 (±0.07) M⁻¹ s⁻¹, respectively.     

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at − 5 °C, and then cooled slowly to − 30 °C using low cooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation–dehydration, vitrification, encapsulation–vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants.     

Purification, Characterization, and Crystallization of anN-HydroxyarylamineO-Acetyltransferase fromSalmonella typhimurium

TheN-hydroxyarylamineO-acetyltransferase fromSalmonella typhimuriumhas been expressed as a histidine-tagged fusion protein inEscherichia coliand purified to apparent homogeneity using single-step immobilized metal ion chromatography. Sufficient quantities of the purified protein have been obtained to allow its characterization by physical methods including dynamic light scattering and electrospray mass spectrometry. The substrate specificity and temperature sensitivity of the enzymatic activity have also been assessed. The enzyme has been crystallized from sodium, potassium tartrate and X-ray diffraction data have been obtained to allow the identification of an orthorhombic unit cell, point group P2₁2₁2, with dimensionsa= 137 Å,b= 223 Å, andc= 105 Å. These crystals will provide a route to a crystallographic determination of the structure of the protein.     

First records of flower biology and pollination in African Annonaceae: Isolona, Piptostigma, Uvariodendron, Monodora and Uvariopsis

Seven species from five genera of Annonaceae were studied with regard to their flower biology and pollination in the Southwest Province of Cameroon, West Africa. They have protogynous hermaphroditic flowers, with exception of Uvariopsis species, which are monoecious. Fused petals of Isolona campanulata remain apically spreading and open during anthesis but form a deep basal urceolate tube around the reproductive organs. At anthesis the yellow pendent flowers emit a fruit-like scent and attracted small beetles, the likely pollinators. Piptostigma sp. flowers also emit a fruit-like scent but provide a closed pollination chamber formed by the three inner petals. Small staphylinid beetles attracted during the female stage of anthesis are released from the flowers at the end of the male stage 2-3 days later. Both species have diurnal anthesis, attracting and releasing the flower visitors during daytime. In contrast, Uvariodendron connivens and U. calophyllum have nocturnal anthesis with floral thermogenesis, produce spicy, aromatic and fruity scents and attract large Scarabaeidae beetles, the pollinators, along with many curculionid beetles, which were principally predators of the thick petals. The very large flowers of Monodora tenuifolia have yellowish petals which are spotted with dark red markings. Together with the sweetish, slightly disagreeable scent the flowers attract flies, principally dung flies. The two investigated Uvariopsis species are monoecious with pistillate and staminate flowers being functional at the same time. The violet red flowers of U. bakeriana visually seem to mimic the fruiting body of certain stinkhorn fungi (Phallaceae) although without producing their strong unpleasant carcass stench. Flower-visiting dung flies were rare. Conversely, U. congolana has a strong fungus-like scent, its flowers are presented at litter height and dung flies living in the litter were the flower visitors, albeit sporadic. The 4-5 days lasting anthesis of both Uvariopsis species appears to be an evolutionary consequence of their diffuse pollinator spectra. The studied African Annonaceae therefore have either cantharophilous or myiophilous/sapromyiophilous flowers with, in part, respectively, remarkably long anthesis, thermogenesis, and widely open, large flowers - all attributes unknown or rare in the hitherto better studied Neotropical Annonaceae.     

Synthesis,Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.