Determination of pharmacokinetics of 8-chloroadenosine and its two major metabolites in dogs by high-performance liquid chromatography

High-performance liquid chromatography was used to measure concentration of 8-chloroadenosine (8-Cl-A) and its two major metabolites 8-chloroadenine (8-Cl-Ad) and 8-chloroinosine (8-Cl-I), and their pharmacokinetics in dogs. 8-Cl-A and its metabolites in serum were treated by deproteinization with acetonitrile, then organic impurities were extracted with dichloromethane, followed by centrifuged and direct injection of the supernatant into the liquid chromatograph. After intravenous injection of 8-Cl-A (30 mg/kg), the parent drug and 8-Cl-I were not detected, but the other metabolite, 8-Cl-Ad, was found at a high concentration for 240 min in dog serum. The main pharmacokinetic parameters of 8-Cl-Ad, t_1/2β and AUC, were 69.30 min and 580 μg min/ml. Our finding indicates that in dogs 8-Cl-A is rapidly metabolized and forms its major metabolites, 8-Cl-Ad and 8-Cl-I. 8-Cl-Ad appeared in many tissues, but 8-Cl-A and 8-Cl-I did not. The concentration of 8-Cl-Ad in dog tissues was highest in the liver and spleen, intermediate in the kidney, intestine, and lowest in the bone marrow, heart, and lungs. However, it was not detected in some liposoluble tissues such as the testes, brain, or uterus. Our study provides useful information for clinical experiment.     

Induction and repair of psoralen cross-links in DNA of normal human and xeroderma pigmentosum fibroblasts

Skin fibroblasts from normal human subjects were exposed in vitro to long-wave ultraviolet radiation (UVA, 320–400 nm) alone, or in combination with 8-methoxypsoralen (8-MOP). DNA damage was analysed with the alkaline elution technique before and after post-treatment incubation of the cells at 37°C for various times.Cells treated with UVA at 1.1 J/cm² showed an increased DNA elution rate, which returned to the normal level within 30 min of post-treatment incubation. In cells treated with PUVA (8-MOP at 20 μg/ml plus UVA at 0.04 J/cm²), the alkaline elution rate was not different from untreated control cells, either before or after post-treatment incubation for times up to 7 days.When the PUVA treatment was followed first by a washing, to remove any unbound 8-MOP, and then by UVA (PUVA + UVA) at 1.1 J/cm², the alkaline elution rate decreased below the control level. During the post-treatment incubation of the PUVA + UVA-treated cells there was a gradual increase of the alkaline elution rate to a level significantly above that in control cells. This increase was observed after 30 min. It reached a miaximum after 24 h and remained after 7 days of post-treatment incubation. Cells from a patient with xeroderma pigmentosum of complementation group A, which were given the same PUVA + UVA treatment, did not show any change in the alkaline elution rate during the post-treatment incubation.If, as seems likely, an increased alkaline elution rate indicates an increase of DNA breaks, and a decreased alkaline elution rate indicates the sealing of breaks and/or the formation of cross-links, the results would suggest the following: (1) UVA irradiation in itself is capable of inducing DNA breaks, which are rapidly sealed during post-treatment incubation; (2) PUVA treatment induces mono-adducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation and can be activated to form DNA cross-links by a second dose of UVA; (3) DNA cross-links induced by PUVA + UVA can be recognized by a repair process that involves the formation of DNA breaks. This process is not observed in xeroderma pigmentosum cells of group A.     

Mass Spectra and Fragmentation of Some 2-Substituted 9-(o-Chlorobenzyl)-8-Azahypoxantines

The regularities of mass spectrometric fragmentation under electron impact of new 9-(-chlorobenzyl)-8-azahypoxantines with (N-aryl)amidocarbonylmethylthiomethyl substituents in position 2 were studied. The main fragmentation pathways are the elimination of Ar-NH⁺ and -chlorobenzyl ions and cleavage of C–S bonds, characteristic of organic sulfides. During the fragmentations, some rearrangements occur, consisting in the transfer of labile hydrogen atoms from the -positions to ions being eliminated. Fragmentation of 8-azapurine parts of the molecules does not prevail. Peaks of molecular ions are clearly visible in the mass spectra of all the substances studied.     

New 8-hydroxyflavonoids from Solanum section Androceras

Five new 8-hydroxyflavonoids have been identified from leaves of Solanum section Androceras: 8-methoxymyricetin 3,7,4′-trimethyl ether; 8-hydroxymyricetin 3,7,4′-trimethyl ether; 8-hydroxymyricetin 8-O-glucosylxyloside 3,7,4′-trimethyl ether; 8-hydroxychrysoeriol 7-methyl ether; 8-hydroxychrysoeriol 7-O-glucoside.     

轮状病毒vp8基因在原核系统中的初步表达

通过RT-PCR反应获得轮状病毒Wa株 vp8基因的cDNA片段,将其克隆入pGEX-5X-1表达载体中,构建重组质粒pGEX-VP8,转化大肠杆菌JM109,筛选阳性克隆子并对插入片段vp8进行序列测定,诱导后通过SDS-PAGE检测重组蛋白,并观察表达量随时间变化的特征.结果显示,测序结果与vp8序列一致,VP8蛋白的表达量在诱导后6-8h达到高峰.     

人类胚胎干细胞分化前后CDCA8基因启动子区甲基化状态的实验研究

目的：分析在人类胚胎干细胞分化过程中,CDCA8基因启动子区甲基化的状态.方法：生物信息学预测人类CDCA8基因上游2 kb区域的CpG岛.抽提未分化和自然分化的人类胚胎干细胞gDNA,应用重亚硫酸盐修饰和DNA序列分析方法检测CDCA8基因启动子区CpG岛甲基化情况.结果：未分化和自然分化的人类胚胎干细胞中,被检测的CDCA8基因启动子区CpC岛均未发现明显的甲基化修饰.结论：在人类胚胎干细胞分化前后,CDCA8基因启动子关键区域的甲基化状态未发生明显改变.     

深海嗜热噬菌体GVE2头尾连接蛋白EV8的原核表达和功能分析

为了解头尾连接蛋白在噬菌体感染和装配中起的作用,对高温噬茵体GVE2(virulent bacteriophage of Geobacillus sp.E263)的头尾连接蛋白EV8进行了原核表达和功能鉴定.将EV8编码序列克隆入pGEX4T-2原核表达载体,并转染大肠埃希氏菌BL21.诱导表达后用SDS-PAGE进行鉴定,显示获得相对分子质量约36kD的谷胱甘肽硫转移酶(GST-EV8)融合蛋白.Western blot检测发现EV8在GVE2感染后2h开始表达,说明该蛋白为噬菌体晚期表达蛋白.免疫电镜定位表明头尾连接蛋白位于噬菌体头尾的连接处,为进一步研究高温噬菌体的组装和表达调控奠定了基础.     

CK8寡糖链结构及翻译水平改变与人肝癌细胞转移相关性研究

糖组学方法筛查人肝癌细胞转移过程中发挥重要作用的核心岩藻糖基化蛋白质分子,解析比较筛出的差异蛋白——细胞角蛋白8(CK8)翻译及糖基化修饰改变与人肝癌细胞转移潜能的关系.应用双向电泳(2-DE)、凝集素亲和印迹、凝集素亲和沉淀联合质谱分析技术,筛查并验证与肝癌转移相关的核心岩藻糖基化蛋白;应用细胞免疫荧光和蛋白质免疫印迹检测CK8的蛋白质表达情况;应用免疫沉淀结合多种凝集素亲和印迹,推测其与肝癌转移相关的寡糖链结构改变.研究发现,3种不同转移潜能人肝癌细胞Hep3B、MHCC97-L和MHCC97-H的扁豆凝集素(LCA)亲和印迹表达谱中,分子质量55～60ku、等电点4～6区域处有核心岩藻糖基化蛋白呈差异表达,质谱鉴定为CK8.LCA亲和沉淀及蛋白质印迹进一步验证CK8异常核心岩藻糖基化与肝癌转移相关;研究发现,CK8分布于细胞浆内,在MHCC97-L和MHCC97-H细胞中蛋白质表达水平较Hep3B高,在MHCC97-H中与LCA和蓖麻凝集素(RCA-1)的亲和力较Hep3B强.以上结果提示,2-DE和凝集素印迹技术联合MALDI-TOF-MS/MS分析可用于筛查疾病过程相关的异常糖基化蛋白质分子,CK8蛋白水平、核心岩藻糖基化及β-1,4末端半乳糖基化的增加均与肝癌细胞转移潜能相关.     

Detection of 8-hydroxydeoxyguanosine in K562 human hematopoietic cells by high-performance capillary electrophoresis

A method for the detection of 8-hydroxydeoxyguanosine by high-performance capillary electrophoresis (HPCE) was developed. Separations were performed in an uncoated silica capillary (44 cm × 75 μm I.D.) with a P/ACE system with diode-array detector. The separation of purine deoxynucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.5), 25°C, 25 kV, 20 s load and detection at 254 nm. This method allowed the detection of 8-hydroxydeoxyguanosine in the presence of a 10⁵-fold higher amount of deoxyguanosine. Isolated nuclei from K562 human hematopoietic cells were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated, enzymatically hydrolyzed to the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxide treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosine than untreated controls. HPCE analysis of 8-hydroxydeoxyguanosine is fast and simple. Furthermore, it requires a very small sample volume, which makes it useful for biomedical and clinical applications.     

8-OH-DPAT对大鼠体感皮质5-羟色胺抑制单位自发放电的抑制作用

目的：研究5-羟色胺（5-HT）对大鼠大脑皮质第一体表感觉区自发单位放电（SI-SUD）的影响,以及5-HT1A受体在5-HT抑制SI-SUD中的可能作用。方法：记录微电泳5-HT及5-HT1A受体选择性激动剂8-OHDPAT前后的SISUD,分析：MISISUD的平均放电间隔（MISI）变化并作统计学处理。结果：①微电泳5-HT对SISUD的影响有3种情况：则MISI增大（抑制作用）（48／96）;MISI减小（兴奋作用）（26／96）或MISI无明显改变（无明显影响）（22／96）。其中以抑制作用为主。②在20个5-HT押制单位中,微电泳8-OH-DPAT可抑制其中17个单位的SISUD,而其余3个单位的SISUD无明显改变。结论：5-HT对SISUD的影响以抑制作用为主。体感皮质的大部分5-HT抑制单位存在有5-HT1A受体,并参与5-HT对SI-SUD的抑制作用。     

光温条件对光温敏核不育小麦ES-8不育基因表达的影响

采用分期播种试验方法,研究了光温条件对光温敏核不育小麦ES8不育基因表达的影响。结果表明,光温条件的变化影响不育基因的表达,在长沙地区,ES8在9月5日至10月15日播种,花粉败育率达100%,不育株率达100%.在12月5日至12月15日播种,其自交结实率为71.6～72.7%,在10月25日至11月25日播种,表现为部分可育和部分不育。ES8存在两个育性转换的临界期,10月25日播期为不育变为半不育的育性转换临界期,12月5日为部分可育变为完全可育的育性转换临界期。育性转换的临界日长约小于11.50h.     

MutY is Down-regulated by Oxidative Stress in E. coli

In Escherichia coli, MutM (8-oxoG DNA glycosylase/lyase or Fpg protein), MutY (adenine DNA glycosylase) and MutT (8-oxodGTPase) function cooperatively to prevent mutation due to 7, 8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic oxidative DNA adduct. MutM activity has been demonstrated to be induced by oxidative stress. Its regulation is under the negative control of the global regulatory genes, fur, fnr and arcA. However, interestingly the presence of MutY increases the mutation frequency in mutT- background because of MutY removes adenine (A) from 8-oxoG:A which arises from the misincorporation of 8-oxoG against A during DNA replication. Accordingly we hypothesized that the response of MutY to oxidative stress is opposite to that of MutM and compared the regulation of MutY activity with MutM under various oxidative stimuli. Unlike MutM, MutY activity was reduced by oxidative stress. Its activity was reduced to 30% of that of the control when E. coli was treated with paraquat (0.5 mM) or H₂O₂ (0.1 mM) and induced under anaerobic conditions to more than twice that observed under aerobic conditions. The reduced mRNA level of MutY coincided with its reduced activity by paraquat treatment. Also, the increased activity of MutY in anaerobic conditions was reduced further in E. coli strains with mutations in fur, fnr and arcA and the maximum reduction in activity was when all mutations were present in combination, indicating that MutY is under the positive control of these regulatory genes. Therefore, the down-regulation of MutY suggests that there has been complementary mechanism for its mutagenic activity under special conditions. Moreover, the efficacy of anti-mutagenic action should be enhanced by the reciprocal co-regulation of MutM.     

Gas8蛋白的抗体制备及小鼠组织表达图谱分析

目的：制备Gas8抗体,并检测小鼠各组织中Gas8表达谱。方法：在大肠杆菌BL21（DE3）中表达His标签标记的Gas8融合蛋白,以纯化的Gas8重组融合蛋白为抗原制备兔Gas8多抗血清,进一步利用亲和纯化方法制备Gas8多克隆抗体;为确定Gas8抗体的特异性,通过免疫印迹方法检测瞬时表达的Flag-Gas8融合蛋白,同时以抗Flag单克隆抗体作为对照;利用制备的抗体通过Westernblot检测Gas8在小鼠各组织中的表达谱。结果：与Gas8特异性结合的抗体能够通过免疫法获得;利用制备的抗体检测到Gas8在小鼠心、肺、肾、脑、肝、脾、肌肉、睾丸各组织中均表达。结论：获得了特异性抗Gas8抗体,用该抗体可以检测到小鼠组织中Gas8的表达。     

Convenient Synthesis of 8-Amino-2′-deoxyadenosine

Abstract

We studied the behaviour of 8-azido-2′-deoxyadenosine and 8-bromo-2′-deoxyadenosine in aqueous solutions of ammonia and primary and secondary amines. Unexpectedly, 8-Azido-2′-deoxyadenosine is converted to 8-amino-2′-deoxyadenosine in excellent yields. The use of this reaction for the preparation of 8-aminoadenine derivatives needed for the preparation of oligonucleotides carrying 8-aminoadenine is discussed.     

Free Radical Dna Adduct 8-Oh-Deoxyguanosine Affects Activity of Hp a II and Msp I Restriction Endonucleases

8-OH-deoxyguanosine can diminish the ability of the restriction endonucleases Hpa II and Msp I to cleave DNA. The exact position of the adduct within the recognition site appears to determine the extent of the effect.     

MHC-Ⅰ在特发性炎性肌病患者肌肉组织中表达的研究

目的:探讨MHC-Ⅰ在IIM发生发展中的作用和机制.方法:对收集的15例IIM患者进行肌肉活检,观察其病理结构的改变、MHC-Ⅰ在患者肌肉组织中的分布表达情况,并与非炎症性肌病患者作对照.结果:1.在IIM患者肌肉组织中明确有MHC-Ⅰ类分子的表达,其阳性率为100%;2.虽有6例IIM患者肌纤维中没有明显的炎性细胞浸润,但患者还是出现了临床症状且肌酶异常;3.MHC-Ⅰ在IIM肌肉组织中的表达强弱程度与病人肌酶呈正相关性,且具有统计学意义(P＜0.05).结论:MHC-Ⅰ类分子可以作为IIM早期诊断的指标之一,其表达强弱与患者病情程度有相关性.