从噬菌体抗体库中筛选D二聚体特异的Fab抗体

目的:从人源性噬菌体抗体库中筛选抗人D二聚体单克隆抗体并进行可溶性表达。方法:以人D二聚体标准品为抗原对所构建的噬菌体抗体库进行两轮“吸附-洗脱-扩增”筛选,从中筛选出抗人D二聚体的噬菌体Fab抗体,并在大肠杆菌细胞中进行可溶性表达。结果:经4次电转化构建了库容为2. 8×108cfu的抗体库,滴度为4. 1×1014PFU/mL,Fab基因重组率为46%。经过淘筛后携带抗人D二聚体抗体的特异性噬菌体得到了明显的富集。用试剂盒以ELISA法检测对人D二聚体的结合活性,得到抗人D二聚体单克隆抗体。筛选出的阳性克隆成功在大肠杆菌细胞中可溶性表达筛选出的阳性克隆在大肠杆菌细胞中可溶性表达。结论:人D二聚体标准品对抗体库的淘筛,富集了表面呈现抗人D二聚体的单克隆抗体的噬菌体,成功构建了人源性抗D二聚体噬菌体抗体,并成功在大肠杆菌细胞中可溶性表达。     

An immunochemical method for quantitative determination of latent antithrombin, the reactive center loop-inserted uncleaved form of antithrombin

Antithrombin (AT) is a serine protease inhibitor that has thrombin, factors IXa and Xa as target proteases. In addition to active native AT, two other forms have been identified in plasma: the reactive center loop inserted cleaved and latent, uncleaved forms. Both have been shown to be present in normal human blood. Latent AT forms a dimer with native AT in vitro, thus inactivating the native form. Here we describe a mouse monoclonal antibody, 8C8, that is specific for latent AT. The affinity of 8C8 was found to be 500-fold higher for latent than for native AT and 5000-fold higher for latent than for cleaved AT. A sandwich assay was developed to measure the concentration of latent AT in plasma, which was found to be approximately 4.8 mg L(-1) in healthy individuals. The K(D) of the interaction between native and latent AT was found to be 51 mum, i.e. far above the plasma concentration of both native and latent AT, indicating a negligible complex formation in blood.     

D—二聚体在泌尿系肿瘤中的应用

为了解Ｄ二聚体在泌尿系肿瘤患者中的应用价值，采用定量酶联免疫吸附法对４７例泌尿系肿瘤患者及７３例非肿瘤患者血浆Ｄ二聚体含量进行了测定。结果显示：肿瘤患者血浆Ｄ二聚体含量较非肿瘤患者及正常组明显增高（Ｐ＜０．００１），非肿瘤患者与正常组之间无显著性差异（Ｐ＞０．０５）。结果表明，泌尿系肿瘤患者体内处于继发性纤溶活性增高状态，Ｄ二聚体可作为监测泌尿系肿瘤患者的指标之一。     

Usefulness of Ictal and Interictal 99mTC Ethyl Cysteinate Dimer Single Photon Emission Computed Tomography in Patients with Refractory Partial Epilepsy

Summary: Purpose: Ictal perfusion single photon emission computed tomography (SPECT), using HMPAO, has been shown to localize epileptic foci in ～90% of studies. Unfortunately, HMPAO decomposes rapidly, precluding the performance of ictal studies. Ethyl cysteinate dimer (ECD) is a SPECT perfusion agent recently approved by the Food and Drug Administration. After preparation, this compound is stable for ～6 h. facilitating the performance of ictal studies. Methods: In a prospective, open-label, uncontrolled, non randomized study, we evaluated the potential benefits of the use of ^99mTc-ECD SPECT for lateralization of the epileptic focus. Ten consecutive adult epilepsy surgery candidates were studied with ictal and interictal ^99mTc-ECD SPECT. Results: The mean delay between seizure onset and ictal SPECT injection was 23.2 s. The mean seizure duration was 84.1 s. Ictal studies agreement between the epilepsy focus and area of hyperperfusion was evident in 8 of 10 cases. In one case, SPECT was lateralized in a patient with bilateral temporal lobe epilepsy (TLE); however, hyperperfusion was observed on the same side of that particular seizure. In another case, there was location disagreement. Interictal SPECT showed focal hypoperfusion in three cases. Conclusions: ^99mTc-ECD proved to be an optimal tracer for ictal studies. Although this is a small series, the results of ictal and interictal findings using 99mTc-ECD are similar to those reported with ^99mTc-HMPA0. Because ^99mTc-ECD has a longer decomposition time, true ictal studies are easier to obtain. This new tracer will probably allow the use of ictal SPECT to become widely accepted in most epilepsy centers.     

Synthesis of side-chain to side-chain cyclized peptide analogs on solid supports

Cyclic peptide structures of the type -Lys-R₁—R_n-Glu- can be synthesized on the Merrifield resin by assembling the peptide chain using Nα-Fmoc-amino acids and Boc and tert.-butyl protection for the side-chains of Lys and Glu, respectively. If residues R₁ to R_n contain side-chain functional groups, TFA-resistant protection is required. After TFA treatment cyclization on the resin can be performed with appropriate coupling reagents. The formation of such cyclic structures may be preceded or followed by peptide chain assembly using Nα-Boc-amino acids and the entire peptide chain containing the cyclic portion is finally cleaved by HF treatment. Using this principle we synthesized the following opioid peptide related cyclic analogs: H-Tyr-d -Lys-Gly-Phe-Glu-NH₂ (I), H-Tyr-Lys-Gly-Phe-Glu-NH₂ (II), H-Tyr-d -Lys-Phe-Glu-NH₂ (III), H-Tyr-d -Glu-Gly-Phe-Lys-NH₂ (IV), H-Tyr-d -Glu-Phe-Lys-NH₂ (V), H-Tyr-d -Orn-Gly-Glu-NH₂ (VI) and H-Tyr-d -Ala-Lys-Phe-Glu-NH₂ (VII). Cyclic monomers were obtained in all cases, as demonstrated by mass spectrometry. Analysis of side-products revealed a slow-down of the HF deprotection of O-benzylated tyrosine as a consequence of hydrophobic interactions as well as the formation of a side-chain-linked antiparallel cyclic dimer in the case of compound VI. In conclusion, the described method permits the convenient preparation of peptide analogs cyclized via amide bond formation between side-chain amino and carboxyl groups in reasonable yield.     

Effects of Ginkgo biloba on haemostatic factors and inflammation in chronic peritoneal dialysis patients

Ginkgo biloba extract decreased plasma D-dimer concentration, a marker of intravascular coagulation, in chronic peritoneal dialysis patients. Blood levels of fibrinogen, von Willebrand factor, hs-CRP, albumin and liver enzyme levels were not significantly changed. No bleeding episode was reported. These results suggest that Ginkgo biloba extract was effective in partially reversing the thrombogenic coagulation profile without increasing the risk of bleeding.     

血D-二聚体、纤维蛋白原、同型半胱氨酸测定在糖尿病肾病中的意义

目的 探讨血D-二聚体（DD）、纤维蛋白原（FIB）、同型半胱氨酸（Hcy）测定在早期糖尿病肾病中的临床意义.方法 选择已确诊的2型糖尿病患者141例,健康对照组46例,测定清晨空腹血DD、FIB、Hcy水平.并同时收集糖尿病患者24h尿液进行尿微量白蛋白（mALB）测定,按照其24h尿白蛋白排泄率分为正常蛋白尿组（mALB≤30mg/24h）及微量蛋白尿组（mALB30-300mg/24h）.结果 2型糖尿病患者DD、FIB在正常蛋白尿组已升高;微量蛋白尿组三项指标均显著高于对照组,差异具有统计学意义（P〈0.01）,且三项联合检测阳性率明显高于单项检测.结论 联合检测DD、FIB、Hcy水平对观察糖尿病患者病情发展变化及提高糖尿病早期肾脏损伤的检出率具有重要的临床意义.     

Using the laboratory to predict recurrent venous thrombosis

Characterisation of heritable thrombophilic defects has facilitated an understanding of the complex mechanisms influencing risk of venous thromboembolism. In parallel with this, the importance of gene-environment interaction in the development of this disease has become apparent. However, testing for a limited number of heritable thrombophilic defects (first generation thrombophilia testing) has not been shown to predict likelihood of recurrent venous thrombosis to any useful degree. This paradox whereby thrombophilia testing identifies defects associated with an increased risk of a first venous thrombosis but not of a particularly high risk of recurrence is likely the result of limitations imposed by a limited dichotomous testing strategy compounded by test inaccuracy and imprecision. Consequently, the observed intermediate phenotype (defined by limited dichotomous testing) is not concordant with the risk of recurrent venous thrombosis. Whilst a simple dichotomous testing strategy for a limited number of heritable thrombophilic defects has not been shown to have useful clinical predictive value, proof-of-principle is emerging for testing of multiple genetic factors in predicting the likelihood of recurrent thrombosis. In addition, recent studies indicate that measurement of the global activity of the coagulation system using either biomarkers or measuring the thrombin generating potential (second generation thrombophilia testing) may have useful clinical predictive value for recurrent thrombosis. The assessment of the intermediate phenotype by global coagulation tests and genome-wide mutation and SNP (single nucleotide polymorphisms) detection may provide complimentary approaches to the quantification of risk of recurrence and enable a move towards more patient-focussed rather than disease-focussed care.     

Cross-Receptor Interactions between Dopamine D(2L) and Neurotensin NTS(1) Receptors Modulate Binding Affinities of Dopaminergics

Dopaminergic systems have been described to functionally interact with the neuromodulatory peptide neurotensin. Employing fluorescence detected coimmunoprecipitation and radioligand binding experiments, we herein demonstrate that coexpression of dopamine D(2L) receptor and the neurotensin receptor subtype NTS(1) leads to physical interaction and the formation of heteromers in transfected human embryonic kidney 293 cells. In this in vitro system, a trans-inhibitory effect on the agonist binding affinity of D(2) was observed in presence of neurotensin. To correlate between the functional properties of dopaminergic agents and the magnitude of neurotensin-induced modulation of D(2L) binding affinities in cells coexpressing D(2L) and NTS(1), a structurally diverse set of dopamine receptor agonists, partial agonists, and antagonists was tested. Ligand specific profiles indicating substantial bias between ligand efficacy and transmodulation were discovered, suggesting a heteromerization-based functional selectivity. In the presence of neurotensin, the novel D(2) agonist FAUC 326 displayed a 34-fold decrease of binding affinity in cells coexpressing D(2L) and NTS(1).     

D二聚体对子痫前期的预测价值

目的分析孕妇第10至15周血浆D-二聚体能否作为预测子痫前期的指标。方法回顾性地分析了85例正常孕妇、47例轻度子痫前期孕妇和27例重度子痫前期孕妇的病历,采用受试者工作特征曲线分析法评价了妊娠第10～15周的血浆D-二聚体水平对子痫前期的预测价值。结果发生子痫前期的孕妇血浆D-二聚体水平高于正常孕妇（P〈0．01）,重度子痫前期孕妇血浆D-二聚体水平高于轻度子痫前期孕妇。血浆D-二聚体水平预测子痫前期的曲线下面积（AUC）为0．71（95％可信区间为0．63—0．79）。当D-二聚体取值为396．5μg／L时,对应的预测敏感性（95％可信区间）和特异性（95％可信区间）分别为0．58（0．46～0．69）、0．81（0．71—0．89）,预测优势比（OR）为5．98。结论妊娠第10～15周血浆D-二聚体水平对预测子痫前期具有一定帮助。