NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite acyclic lesions

Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated APP 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated APE 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the APP 9-mer and APE 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution between -5 and 5 degrees C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and E14) and stacks between the flanking G4.C15 and G6.C13 Watson-Crick base pairs in both the APP 9-mer and APE 9-mer duplexes. The helix is right-handed at and adjacent to the abasic site, and all glycosidic torsion angles are anti in both 9-mer duplexes. Proton NMR parameters for the APP 9-mer and APE 9-mer duplexes are similar to those reported previously for the APF 9-mer duplex (F = furan) in which a cyclic analogue of deoxyribose was embedded in an otherwise identical DNA sequence [Kalnik, M. W., Chang, C. N., Grollman, A. P., & Patel, D. J. (1988) Biochemistry 27, 924-931]. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the APP 9-mer and APE 9-mer duplexes (5 degrees C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles. These same torsion angles are also distorted in the APF 9-mer but assume a different conformation than those in the APP 9-mer and APE 9-mer duplexes.     

Study on the variables affecting toxicity of hybrid toxins. The effect of different target cell receptor distribution and toxin binding chain

Disulfide conjugates of diphtheria toxin (DT) and its fragment A (DTA) to asialoorosomucoid (ASOR) were prepared. The toxicity of the conjugates were compared with DT in isolated rat, rabbit and guinea pig hepatocytes containing different concentration of asialoglycoprotein receptors (Biochim. Biophys, Acta 942, 57, 1988). In rat hepatocytes DTA-ASOR was highly toxic with half-maximal inhibitory concentration (IC50) of protein synthesis occurring at 4 +/- 3.10(-11) M (n = 7) which was much lower than that of DT DT (7.8 +/- 9.8.10(-9) M, n = 7). In rabbit hepatocytes toxicity of the conjugate (IC50 = 5.4 +/- 4.9.10(-10) M, n = 7) was higher than that of DT (IC50 = 5 +/- 4.10(-11) M, n = 7). In guinea pig hepatocytes, DTA-ASOR was not toxic at concentration below 10(-8) M, although DT was highly toxic (IC50 = 1.8 +/- 1.4.10(-10), n = 3). In the presence of 5 microM colchicine, the toxicity of DTA-ASOR in rat and rabbit hepatocytes increased by 10-fold, while in guinea pig hepatocytes it became detectable with an IC50 of 1.2 +/- 0.8.10(-9) M (n = 3). The toxicity of DT in the rat cells was also enhanced 10-fold by colchicine, but not at all in either the rabbit or the guinea pig cells. Addition of isolated diphtheria toxin fragment B (DTB) did not affect significantly the toxicity of DTA-ASOR in all three hepatocytes and that of DT in rat hepatocytes, but reduced toxicity of DT more than 20-fold in the rabbit and guinea pig cells. Toxicity of DT-ASOR in rat hepatocytes was the same as DTA-ASOR both in the absence and presence of colchicine, and abolished completely by excess ASOR, but not by DTB. Toxicity of DT-ASOR in rabbit hepatocytes was 40-times higher than DTA-ASOR, enhanced 10-fold by cochicine and reduced more than 30-fold by excess ASOR, but only slightly by DTB. These results indicate that entry of DTA from DTA-ASOR involve a DTB-independent translocation mechanism which can be as efficient as the DTB-dependent mechanism used by DT in the rabbit and guinea pig cells. The entry of both conjugates appeared to be mediated by the asialoglycoprotein receptors. However, the DTB moiety of DT-ASOR could function only in the DT-sensitive cells indicating the lack of a DTB-mediated translocation in the DT-resistant cells.     

Deficient glycosylation of arylsulfatase A in pseudo arylsulfatase-A deficiency

Summary Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.Abbreviations PD Pseudo arylsulfatase-A Deficiency - ARA Arylsulfatase-A     

Conversion of versiconal acetate to versiconal and versicolorin C in extracts from Aspergillus parasiticus

The primary product of hydrolysis of versiconal acetate catalyzed by porcine liver esterase and the 35–70% ammonium sulfate fraction from a soluble extract from mycelia of Aspergillus parasiticus was versiconal. Versiconal was stable at neutral pH for several hours and was rapidly converted to versi-colorin C by treatment with 0.4 M HCl. The addition of NADPH to the 35–70% ammonium sulfate fraction resulted in conversion of versiconal acetate to both versiconal and versicolorin C. The conversion of versiconal acetate to versicolorin C in the cell-free system is proposed to involve an esterase and an NADPH-dependent cyclase.     

辣椒素引起脊髓P物质释放及其对血压的影响

为进一步研究脊髓 P 物质(SP)在调节心血管活动中的作用,在大鼠脊髓蛛网膜下腔注射(ith)辣淑素(cap),以刺激脊髓 SP 能神经末梢释放 SP,结果引起血浆去甲肾上腺素(NA)和肾上腺素(AD)含量增高,及具有剂量依赖性的动脉血压上升,心率升高。ith 具有高度特异性的 SP 受体拮抗剂或 SP 抗血清均可阻断 cap 引起的升压效应,免疫组化测定也观察到注入的cap 剂量越大,脊髓胸段 SP 样免疫阳性反应物的致密度越低,这些观察结果支持 cap 可以引起脊髓内 SP 的释放的说法。在第一颈段(C_1)横断脊髓后 ith cap 所引起的升压效应与完整动物 ith cap 的升压效应无显著差异。以上结果提示脊髓 SP 能神经末梢释放的 SP 可以通过交感肾上腺髓质系统引起心血管兴奋效应,SP 可能是引起交感节前神经元兴奋的神经递质。     

大鼠隔—海马通路损伤对海马内递质含量及酶活力的影响

单侧切断大鼠海马缴和部分穹窿可使海马部分去神经。损伤后7d,海马内胆碱能系统中乙酰胆碱(ACh)含量下降72.5%,胆碱乙酰基转移酶(ChAT)活力下降45.7%,胆碱酯酶活力下降52.2%,在单胺能系统中,去甲肾上腺素(NA)含量下降16.3%,多巴胺(DA)含量下降31.3%,5-羟色胺含量下降30.3%。在损伤过程中,海马内氨基酸含量没有改变。实验结果表明,海马缴和穹窿是脑内胆碱能和单胺能传入神经到达海马靶区的部分共同通路。     

去甲肾上腺素、乙酰胆碱对绵羊心肌浦肯野纤维瞬时性内向离子流的影响

用电压箝制术观察了去甲肾上腺素、乙酰胆碱对绵羊浦肯野纤维由乙酰毒毛旋花子甙元诱发的瞬时性内向离子流(I_Ti)的效应。当乙酰毒毛旋花子甙元浓度为4.5×10~(-8)mol/L 时,诱发出的I_(T1)稳定并能维持约1.5h。去甲肾上腺素1.5×10~(-6)mol/L,可使I_(Ti)的峰值由11.4±2.5nA 增加到14.5±4.1nA(n=11,P相似文献   

In vitro effect of actinomycin D on human neutrophil function

The effect of actinomycin D (ACT-D) on human neutrophil chemotaxis, chemiluminescence (CL), superoxide (O2-) production, phagocytic uptake, and intracellular bacterial killing has been examined. The viability of the ACT-D-treated neutrophils was 98% even at a concentration of 10 micrograms/ml for 4 hr. Using fMLP as the chemotactic factor, depressed chemotaxis was demonstrated following ACT-D (1-10 micrograms/ml) pretreatment of neutrophils as compared with the non-treated controls. Similar ACT-D pretreatment produced the depressed responses in phorbol myristate acetate-induced CL and superoxide production by neutrophils. Moreover, using heat-inactivated human serum as an opsonin for Salmonella enteritidis (NCTC 6676), there was a significant difference in intracellular killing (P less than 0.01) but no difference in phagocytic uptake between ACT-D-treated and non-treated neutrophils. These studies indicate that ACT-D profoundly impairs both intracellular bacterial killing by human neutrophil through an effect on respiratory burst activity and directed cell migration of human neutrophils.     

高山毛顶蛾新亚种及其生物学观察(鳞翅目:毛顶蛾科)

毛顶蛾科Eriocraniidae属较原始的小蛾类群。世界已知19种及1亚种,采自全北区桦树适生带,北纬45°—70°N之间,以北美、欧洲的种类最丰富。亚洲所知甚少,惟日本记载过广泛分布于全北区的2种,即:Erioerania sparrmannella(Bosc)和E.semi-purpurella(Stephens)。我国迄至1980年,才由杨集昆先生描述了发生在华北的这2个广布种。现增记青海产一新亚种,标本保存在青海省农林科学院林业研究所。