9-(2-膦酰甲氧乙基)腺嘌呤及其位置异构体3-(2-膦酰甲氧乙基)腺嘌呤的合成和抗病毒活性研究

Phosphonylmethoxyethyl)adenine (1),PMEA,an acyclic nucleotide withbroad-spectrum antiviral activity was synthesized with some modifications of Holy's procedure.Simutaneously,an N-3 regioisomer(2)of PMEA and a by-preduct, formaldehyde di-[2-(9-adenyl)ethyl] acetal(7)were seperated by silica gel chromatography in the ratio of 50:10:1.Compound(2)and(7) are new compounds that we have not yet found in literatures. The structure of them weredetermined with ¹HNMR,2DNMR, MS and Spot test.Antiviral test showed that N-3 isomer(2)completely lost activity against both HIV-1 and HSV-1 in vitro. It seems that regiospecificity of theacyclic nucleotide structure is important for antiviral activity.     

骨形态发生蛋白4转基因治疗大鼠的胫骨缺损

目的:观察基因工程技术构建人骨形态发生蛋白4复制缺陷腺病毒的成骨效果。方法:实验于2006-03/08在安徽医科大学第一附属医院实验动物中心完成。实验分组:选取普通级雄性SD大鼠30只,体质量(200±10)g,全部动物胫骨上端部分分别造成8mm×5mm长方形缺损。采用自身对照法,右侧骨缺损为实验组,左侧骨缺损为对照组。实验组植入人骨形态发生蛋白4复制缺陷腺病毒复合明胶海绵,对照组植入单纯明胶海绵。实验评估:术后分别于4,6,8周麻醉后处死10只动物,取材行X线、组织病理、免疫组织化学、透视电镜检查,观察成骨情况。结果:纳入30只大鼠,全部进入结果分析。①大鼠胫骨缺损X线、组织病理学检查结果:术后8周实验组和对照组骨缺损均得到修复,但实验组无论从成骨时间、成骨效果、新生骨量等方面都要优于对照组。其中各时间点实验组骨密度明显高于对照组,差异有显著性意义[4周:(95.91±16.33),(87.93±11.52);6周:(128.34±10.64),(102.41±9.81);8周:(138.36±10.49),(121.56±9.63);P<0.01]。各时间点实验组新生骨占骨缺损面积比明显高于对照组,差异有显著性意义[4周:(41.39±5.65)%,(26.58±5.62)%;6周:(80.35±7.25)%,(65.41±6.52)%;8周:(96.45±2.76)%,(82.22±7.30)%;P<0.01]②术后4周免疫组织化学染色结果:实验组软骨及骨痂内呈强阳性反应,而对照组骨痂内骨形态发生蛋白4表达微弱。结论:人骨形态发生蛋白4重组腺病毒具有良好的成骨活性,骨形态发生蛋白4直接转基因治疗能够加快骨缺损的修复。     

Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry

BACKGROUND: The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (r2 = 0.86) and superior precision with a CV < 15 percent for blood samples with > 0.1 percent fetal cells. Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS: The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies.     

Quantitative liver function in patients with rheumatoid arthritis treated with low-dose methotrexate: a longitudinal study

The objectives were to determine quantitative liver function prospectively in patients with rheumatoid arthritis (RA) treated with low-dose methotrexate (MTX), to search for risk factors for a loss of quantitative liver function and to assess the relationship between quantitative liver function and histological staging. A total of 117 patients with RA (ACR criteria, 85 women, mean age 59 yr) had measurements of galactose elimination capacity (GEC), aminopyrine breath test (ABT) and liver enzymes [aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (AP), 7-glutamyl transferase (GGT), bile acids, bilirubin, albumin] before treatment with weekly i.m. MTX injections and every year thereafter. In 16 patients, liver biopsies were performed. Before the introduction of MTX, mean GEC was 6.6 mg/min/kg [5th to 95th percentile (5-95 PC) 5.1- 8.5; reference range 6.0-9.1] and mean ABT was 0.80% kg/mmol (5-95 PC 0.42-1.30: reference range 0.6-1.0). During treatment with MTX [mean weekly dose 11.8 mg (5-95 PC 5.4-20.2), mean observation period 3.8 yr (5-95 PC 0.4-6.9)], significant declines of GEC (-0.12 mg/min/kg per year. t = 3.30, P < 0.002) and ABT (-0.06% kg/mmol per year, t = 4.81, P < 0.001) were observed. Negative correlations were found between the annual change in GEC and GEC at baseline (Rs = -0.40, P < 0.0001), and the annual change in ABT and ABT at baseline (Rs = -0.43, P < 0.0001). No correlations were found between the annual change in GEC or ABT and weekly MTX dose, age or percentage of increased liver enzymes, and no effect of a history of alcohol consumption > 30 g/week became evident. Two patients with Roenigk grade III had impaired quantitative liver function, while 14 patients with Roenigk grades I and II exhibited a high variability of GEC and ABT from normal to abnormal values. The continuous declines in GEC and ABT observed deserve attention in patients with prolonged treatment. Patients with a low GEC or ABT at baseline seem not to be at increased risk for a further loss of quantitative liver function. An impaired GEC or ABT does not necessarily concur with hepatic fibrosis on histological examination.      

The distribution of erythrocyte phospholipids in hereditary spherocytosis demonstrates a minimal role for erythrocyte spectrin on phospholipid diffusion and asymmetry

In the human erythrocyte membrane phosphatidylcholine and sphingomyelin reside mainly in the outer leaflet, whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, are mainly found in the inner leaflet. Maintenance of phospholipid asymmetry has been assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin. To investigate whether spectrin contributes to maintaining the phospholipid transbilayer distribution and kinetics of redistribution, we studied erythrocytes from hereditary spherocytosis patients whose spectrin levels ranged from 34% to 82% of normal. The phospholipid composition and the accessibility of membrane phospholipids to hydrolysis by phospholipases were in the normal range. Spin-labeled phosphatidylserine and phosphatidylethanolamine analogues that had been introduced into the outer leaflet were rapidly transported at 37 degrees C to the inner leaflet, whereas the redistribution of spin-labeled phosphatidylcholine was slower. The kinetics of transbilayer movement of these spin-labeled phospholipid in all samples was in the normal range and was not affected by the level of spectrin. Although these erythrocyte membranes contained as little as 34% of the normal level of spectrin and were characterized by several physical abnormalities, the composition, distribution, and transbilayer kinetics of the phospholipids were found to be normal. We therefore conclude that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.     

A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep.

Genetic mutations with major effects on ovulation rate and litter size in sheep were recently identified in three genes belonging to the TGFbeta superfamily pathway: the bone morphogenetic protein 15 (BMP15, also known as GDF9b), growth differentiation factor 9 (GDF9), and BMP receptor type IB (also known as activin-like kinase 6). Homozygous BMP15 or GDF9 mutations raise female sterility due to a failure of normal ovarian follicle development, whereas heterozygous animals for BMP15 or GDF9 as well as heterozygous and homozygous animals for BMP receptor type IB show increased ovulation rates. In the present work, a new naturally occurring mutation in the BMP15 gene in the high prolific Lacaune sheep breed is described. The identified variant is a C53Y missense nonconservative substitution leading to the aminoacidic change of a cysteine with a tyrosine in the mature peptide of the protein. As for other mutations found in the same gene, this is associated with an increased ovulation rate and sterility in heterozygous and homozygous animals, respectively. Further in vitro studies showed that the C53Y mutation was responsible for the impairment of the maturation process of the BMP15 protein, resulting in a defective secretion of both the precursor and mature peptide. Overall, our findings confirm the essential role of the BMP15 factor in the ovarian folliculogenesis and control of ovulation rate in sheep.     

Blood donation-related neurologic needle injury: evaluation of 2 years' worth of data from a large blood center

BACKGROUND: There is little information in the medical literature on t he clinical spectrum of blood donation-related neurologic needle injury and on its frequency in a blood donor population. STUDY DESIGN AND METHODS: Sixty-six cases of blood donation-related neurologic needle injury were identified from nursing reports made during a 2-year collection period involving 419,000 whole blood donations. Telephone follow-up was completed on 56 of the 66 cases to better define clinical symptoms, the donor's desire for physician consultation, recovery times, and residual effects. RESULTS: Symptoms in 66 donors included numbness or tingling (n = 54), excessive or radiating pain (n = 43), and loss of arm or hand strength (n = 8). Of the 56 donors with complete follow-up, 17 (30%) consulted a physician one or more times. Recovery times in these 56 donors were <3 days (n = 22), 4 to 29 days (n = 17), 1 to 3 months (n = 13) 3 to 6 months (n = 2), and >6 months (n = 2). Fifty-two of 56 donors achieved a full recovery, and 4 other donors had only a mild, localized, residual numbness. The incidence of blood donation-related neurologic needle injury was 1 of every 6300 donations. CONCLUSION: While donor recovery may in some cases require a great deal of time and/or physician consultation(s), total recovery appears to be the rule. The incidence of blood donation-related neurologic needle injury is relatively low.     

人参皂甙诱导的造血细胞内信号传递途径的研究

目的探讨人参皂甙（ＧＳ）刺激造血祖细胞增殖有关的细胞内信号传递途径。方法采用Ｎｏｒｔｈｅｒｎ印迹杂交法、电泳带移动阻滞实验、抗体胶结合移动实验和紫外放射交联实验。结果ＮｏｒｔｈｅｒｎＢｌｏｔ显示经ＧＳ诱导的人巨核细胞株ＣＨＲＦ２８８、Ｍｅｇ０１和ＭＯ７ｅ细胞的ＧＡＴＡ２ｍＲＮＡ水平增高,分别是未经处理细胞的１．８４,２．４３和１．５２倍。但ＧＡＴＡ１ｍＲＮＡ表达水平低,且ＧＳ处理前后也无明显变化。电泳带移动阻滞实验提示ＧＳ诱导的细胞核内ＧＡＴＡ转录调控蛋白与特定的ＤＮＡ序列结合的活性增高。抗体胶结合移动实验证实与ＤＮＡ结合的主要成分为ＧＡＴＡ２蛋白,紫外放射交联实验确定该复合物的相对分子质量约为５０×１０３,符合ＧＡＴＡ２转录调控蛋白。结论ＧＳ诱导细胞内的信号传递途径与转录因子ＧＡＴＡ２有关,ＧＡＴＡ２有介导ＧＳ刺激造血细胞增殖的作用。