3′-Deoxy-3′-fluoroinosine as a potent antileishmanial agent

We studied the antileishmanial activity of 3-deoxy-3-fluoroinosine (3-FI) againstLeishmania tropica andL. donovani. In in vitro cultivation, the EC₅₀ values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3-FI against the promastigotes ofL. tropica andL. donovani were 2.3×10^–7 and 1.0×10^–6 M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC₅₀ value was 1.9×10^–4 M. Leishmania promastigote metabolized 3-FI to 3-deoxy-3-fluoroadenosine 5-triphosphate (3-FATP) but FM3A cells did not. 3-FI was effective againstL. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3-FI (50 mg/kg, given i.v.)showed a cytotoxic effect against the amastigotes ofL. donovani in mice.     

聚合酶3′外切活性对3′硫化修饰引物聚合反应的影响

目的 探讨硫化修饰的次3′末端不配对引物能否引发高保真DNA聚合酶介导的聚合反应的非成熟性终止,即所谓聚合反应的“关”效应。方法 采用配对及非末端不配对的3′硫化修饰引物,研究其对不同保真度DNA聚合酶引物延伸反应的影响。结果 非末端不配对的3′硫化修饰引物也能引发高保真DNA聚合酶介导的聚合反应非成熟性终止,而对低保真DNA聚合酶所介导的聚合反应则无明显影响。同时,3′硫化修饰的配对引物对不同保真度DNA聚合酶引物延伸反应均无影响。结论 硫化修饰的次3′末端不配对引物与3′末端不配对引物对引物延伸的影响相似,同样能引起高保真DNA聚合酶介导的聚合反应的“关”的效应。显然,在单基因遗传病的诊断及单核苷酸多态性(single nucleotide polymorphism,SHIP)的高通量分析等方面,硫化修饰的碱基特异性引物与高保真DNA聚合酶所构成的对SNP敏感的“开／关”系统,具有广阔的应用前景。     

利用3′-RACE法扩增到钙通道基因的3′-端片段

Ｃａ2 是细胞内的第二信使,涉及到细胞的信号传导、细胞运动、分裂、分化、神经递质的合成与释放,以及细胞凋亡等重要的基本生命过程。电压激活的钙离子通道在调节细胞内钙离子水平中起着至关重要的作用。以往报道表明,钙通道基因具有高度的多样性。已克隆的6个钙通道α1...     

Kaposi′s肉瘤一例

Ｋａｐｏｓｉ′ｓ肉瘤一例金红林秋柏患者女，３０岁，广东农民，因一个月前发现全身多处淋巴结肿大，同时伴有胸痛、咳嗽、肝区不适，全身无力，体重下降而于１９９５年１０月１５日来就诊。门诊以恶性淋巴瘤收住院。查体：病人外观虚弱无力，双侧颈部、腋下、腹股沟等区...     

Die seltenen Rh-Phänotypen rhy rh und rh′ rh′ in einer deutschen Familie

Zusammenfassung Nach einer Literaturübersicht wird das Vorkommen der seltenen Rh-Genotypen rr und rr in 2 Generationen einer deutschen Familie beschrieben.     

Mutants from V79 fibroblasts exhibiting hypersensitivity to aphidicolin and 3′-azido-3′-deoxythymidine

One variant, aph^hs-3 was previously isolated based on a hypersensitivity to nontoxic concentrations of aphidicolin, a specific inhibitor of DNA polymerases-alpha and delta. This variant was found to be more sensitive to temperatures above 35°C and to 10 µM of 3-azido-3-deoxythymidine (zidovudine, azidothymidine, or AZT) than the parental 743x cells. DNA polymerase activities in the cell extract or in the partially purified fraction by DEAE-cellulose (DE52) anion exchange column from aph^hs-3 were active at 40°C. No significant differences in deoxynucleoside triphosphate pools were observed at 34°C for both the parental 743x and aph^hs-3 cells. Revertants were isolated at 39°C: six revertants (aph^hs-3-tr1 through aph^hs-3-tr6) were obtained without aphidicolin; one revertant aph^hs-3-tar (the tar clone) was selected in aphidicolin (0.12 µM). The hypersensitivity to aphidicolin (Aph^hs) and AZT (AZT^hs) was cosegregated in the revertant aph^hs-3-tr5 (the tr5 clone), while the tar clone was not AZT^hs. There was a similar increase in the specific activity of³H-labeled DNA in all cell lines after additions of [³H]AZT or [³H]thymidine. Additions of purine or pyrimidine arabinosides (araT, araC, and araA) to all cell lines resulted in a similar cytotoxicity, suggesting the anabolism of dTTP was not defective in the tr5 clone. The spontaneous mutation rate at the hypoxanthine-guanine phosphoryltransferase locus using replating techniques and 6-thioguanine resistance selection was 5×10^–7, 2.2×10^–6, or 1.3×10^–6 per generation for the tr5, 743x, or tar cell lines, respectively. Most importantly, DNA polymerase activities in the cell extract of the revertant tr5 clone were inhibited by 0.5 µM AZTTP. In contrast, no inhibition was observed in those of the parental 743x and revertant tar cells. The cosegregation of both Aph^hs and AZT^hs in the tr5 revertant suggests that these two phenotypes may be a result of the same mutational event.     

Nucleotide sequences and mutations of the 5′-nontranslated region (5′NTR) of natural isolates of an epidemic echovirus 11′ (prime)

Summary.  An echovirus 11′ (prime) virus caused an epidemic in Hungary in 1989. The leading clinical form of the diseases was myocarditis. Hemorrhagic hepatitis syndroms were also caused, however, with lethal outcome in 13 new-born babies. Altogether 386 children suffered from registered clinical disease. No accumulation of serous meningitis cases and intrauterine death were observed during the epidemic, and the monovalent oral poliovirus vaccination campaign has prevented the further circulation of the virus. The 5′-nontranslated region (5′-NTR) of 12 natural isolates were sequenced (nucleotides: 260–577). The 5′-NTR was found to be different from that of the prototype Gregory strain (X80059) of EV11 (less than 90% identity), but related to the swine vesicular disease virus (D16364) SVDV and EV9 (X92886) as indicated by the best fitting dendogram. The examination of the variable nucleotides in the internal ribosomal entry site (IRES) revealed, that the nucleotide sequence of a region of the epidemic 5′-NTR was identical to that of coxsackievirus B2. Five of the epidemic isolates were found to carry mutations. Seven EV11′ IRES elements possessed identical sequences indicating, that the virus has evolved before its arrival to Hungary. The comparative examination of the suboptimal secondary structures revealed, that no one of the mutations affected the secondary structure of stem-loop structures IV and V in the IRES elements. Although it has been shown previously, that the echovirus group is genetically coherent and related to coxsackie B viruses the sequence differences in the epidemic isolates resulted in profound modification of the central stem (residues 477–529) of stem-loop structure No.V known to be affecting neurovirulence of polioviruses. Two alternate cloverleaf (stem-loop) structures were also recognised (nucleotides 376 to 460 and 540 to 565) which seem to mask both regions of the IRES element complementary to the 3′-end of the 18 S rRNA (460 to 466 and 561 to 570), thus probably diminishing initiation of translation. The possible biological importance of the alternative cloverleaf structures is supported by the fact that neither the 17 variable nucleotides nor the two mutations of epidemic isolates within the regions seem to modify the predicted alternative secondary structures in EV11, SVDV and CBV1-4. Accepted May 31, 2000 Received July 21, 1999     

5′和3′末端快速扩增法克隆多发性骨髓瘤负性相关基因DAZAP2

目的在多发性骨髓瘤患者中KIAA0058基因序列表达明显下调,从正常人骨髓单个核细胞中克隆该序列全长cDNA,以期更好地研究该序列结构,获得其功能信息。方法采用末端快速扩增法(RACE),进行多轮RT-PCR后,对PCR产物进行测序、拼接、证实,与NCBI核酸数据库进行比对。结果该序列全长cDNA长度为1913bp,登陆号为AY430097,与来源于人睾丸cDNA文库的序列DAZAP2同源性高达99%以上。该序列具有较短的5′非翻译区及长的3′非翻译区,开放阅读框序列从第84位碱基至590位碱基,具有poly(A)尾。结论利用RACE法,从骨髓单个核细胞中成功克隆了DAZAP2全长cDNA。     

3′碱基特异的PCR技术

引物3′末端的碱基处于一个对引物延伸至关重要的位置。如果该碱基与模板不互补(错配),就可能阻碍引物延伸,在PCR(聚合酶链反应)产物中没有特异长度扩增带。反之,如果该碱基与模板互补,则有特异长度扩增带。作者将引起β地中海贫血的三种最常见突变碱基设计并合成在引物3′末端上,根据PCR产物是否出现特异长度扩增带即可作出模板DNA有无相应突变的判断,而无须任何杂交或酶解。这种PCR称之为3′碱基特异的PCR(3′BS PCR)。     

5′-核苷酸酶活性比色分析

血清及细胞膜上的5′—核苷酸酶活性定量测定法简单,快速、敏感,一般实验室可测,血清中此酶的定量测定能用于肝脏疾病的诊断。因淋巴细胞膜上结合有此酶,所以可用于白细胞亚群的临床检测及培养的淋巴细胞的测定,也能用于白血病及恶性淋巴瘤的研究。据报导,原发性低丙种球蛋白血症(CVK);几种联合性免疫缺陷病;T急性淋巴细胞性白血病、B慢性淋巴性白血病及其他B细胞疾病(例如:B急性淋巴性白血     

Wilson′s病的分子生物学进展

Wilson's病(WD),又称肝豆状核变性,是一种以铜代谢障碍为特征的常染色体隐性遗传病。其致病基因已定位于13q14.3。近年来,通过基因克隆技术已克隆到WD基因,且分离出WD基因的cDNA候选片段Wcl。序列分析及家系突变研究表明,该基因编码与铜离子活化有关的P型ATP酶。基因的突变形式主要为点突变,而且,通过连锁分析开展WD的基因诊断也取得了进展。     

Down′s综合征的产前筛查

目的　通过测定孕中期孕妇血清AFP和 β-HCG的浓度 ,筛查出Down′s综合征高危孕妇 ,并进一步对其诊断以预防Down′s综合征患儿的出生。方法　应用酶联免疫吸附法检测出血清AFP和 β -HCG的浓度 ,结合孕妇年龄、孕周等临床资料利用计算机软件进行统计分析 ,得出结果。结果　在筛查的 12 79名孕妇中 ,筛查出 88例Down′s综合征高危孕妇 ,确诊 1例Down′s综合征胎儿 ,筛查假阳性率为 6 .8%。结论　对孕妇进行胎儿Down′s综合征筛查是可行的 ,但筛查假阳性率较高 ,筛查方法还需进一步探索、改进。     

Ekbom′s综合征一家族报告

Ekbom′s综合征又称不宁腿综合征，在临床上并非罕见，以往曾有家族报告，但合并脑萎缩者未见有报告，现将一Ekbom′s综合征家族报告如下。     

Gaucher′s病二例报告

戈谢病 (Gaucher′sdisease ,GD)是溶酶体贮积性疾病 ,又称葡萄糖脑苷脂贮积病 ,属于常染色体隐性遗传。 1882年由法国Gaucher首次报道。由于染色体lq2 1上葡萄糖脑苷脂酶 (glucocerebrosidase)基因变异 ,导致葡萄糖脑苷脂不能水解而在网状内皮细胞系统的巨噬细胞中大量贮积 ,临床表现为肝脾肿大、贫血、骨骼破坏、发育滞后、反复癫痫、共济失调等。现将我院 2 0 0 4年的 2例GD报告如下。病例 :例 1,女 ,6个月 ,因咳嗽 1月 ,间歇发热 10余天入院。体检 :T 37.2℃ ,P16 0次 /min ,R6 0次 /min ,一般情况较和发育营养较差 ,精神欠佳 ,左腋…     

Adenosine 3′∶5′-cyclic monophosphate mediates a 5-hydroxytryptamine-induced response in neonatal rat motoneurones

5-Hydroxytryptamine (5-HT) is present in nerve fibres descending from the brainstem Raphe nuclei to motoneurones and its release is thought to exert excitatory actions. 5-HT, applied from the outside, directly depolarizes spinal and cranial motoneurones in slices. This action of 5-HT is mediated, in part, by an inwardly rectifying cationic current, I _h. In cardiac cells, an equivalent current, i _f, has been shown to be directly activated by adenosine 35-cyclic monophosphate (cAMP) applied to the inside of the patch membrane. By applying the whole-cell method to thin slices of brainstem and spinal cord, we have shown that intracellularly applied cAMP and extracellularly applied dibutyryl cAMP or forskolin mimics the inward current induced by 5-HT. The selective cAMP phosphodiesterase inhibitor, Ro 20–1724, clearly prolonged the 5-HT-induced current. Maximal doses of dibutyryl cAMP or forskolin occluded the 5-HT-induced current. The broad spectrum protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methlypiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8) had no effect on the currents induced by 5-HT and forskolin. From these results, we suggest that activation of 5-HT receptors on the motoneuronal membrane stimulates formation of intracellular cAMP, thereby inducing the inward current, possibly by a direct action on I _h.     

Suppression of lymphocyte adenosine 3′ : 5′-cyclic monophosphate (cAMP) by delta-9-tetrahydrocannabinol

Delta-9-tetrahydrocannabinol (THC) is the major psychoactive component of marijuana. Suppression of mitogen-stimulated blastogenesis of human lymphocytes in vitro by THC was previously demonstrated. This effect was shown to be concentration dependent with the non-toxic concentrations 5, 7.5, and 10 μg THC/ml showing the greatest suppression. However, the mechanism(s) by which THC induces suppression are still unclear. The current study examines the effect of THC on the adenosine 3′ : 5′-cyclic monophosphate (cAMP) pathway second messenger system, which is involved in activation of human peripheral blood lymphocytes. Lymphocyte cAMP levels were stimulated using three hormone receptor stimulators, isoproterenol, histamine, or 5′-N-ethylcarboxamide adenosine (NECA), each of which utilizes a different receptor to enhance cAMP production. THC suppressed cAMP levels independently of the hormone and receptor utilized. Levels of cAMP in non-mitogen-stimulated peripheral blood mononuclear cells and plastic non-adherent lymphocytes, as well as cells stimulated with phytohemmagglutinin, were suppressed by THC. Suppression of cAMP production by THC was further examined to determine whether inhibition involved a GTP-binding protein (G_i), which is known to down-regulate cAMP production. Cells were pre-treated with pertussis toxin to inhibit G_i activity; this blocked the THC-induced suppression of cAMP production. These results suggest that THC can exert its effects on second messenger systems at the lymphocyte membrane level, and that a pertussis toxin-sensitive G_i protein may be involved. Thus, second messenger regulated pathways may be involved in THC-induced immune suppression. However, the relationship between alteration of cAMP production and suppression of lymphocyte function due to the presence of THC in the medium remains to be established.