铽离子与RNA的荧光反应及RNA测定的研究

核糖核酸（ＲＮＡ）与稀土铽离子在ｐＨ５．０～６．５条件下能形成具有较强荧光的络合物,其激发峰在２８８ｎｍ处,发射峰为４９４ｎｍ及５４５ｎｍ处．ＲＮＡ在０．１～１０ｍｇ／Ｌ范围内与其荧光强度呈线性关系,其检出限为６．０×１０￣（－８）ｍｏｌ／Ｌ．腺苷酸,尿苷酸,胞苷酸对ＲＮＡ的干扰比较小,因此可在其存在下选择性地测定ＲＮＡ．     

粘质赛氏菌胞外蛋白酶的提取、纯化及部分性质研究

经过硫酸铵３０％～５０％分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达５３％,并制备了酶的结晶,该酶以ＳｅｐｈａｄｅｘＧ１００柱层析及ＳＤＳ－ＰＡＧＥ测得分子量约为８１０００,该酶的最适ｐＨ为７．０,最适温度为４５℃,Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋、Ｃｕ２＋、Ｃｏ２＋等重金属离子不同程度地抑制酶活性。     

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt II sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine- or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.Abbreviations ELISA enzyme-linked immunosorbent assay - NOS nopalene synthase - nos DNA segment encoding NOS - NPT II neomycin phosphotransferase - npt II DNA segment encoding NPT II - PAGE polyacrylamide gel electrophoresis     

大鼠不同脑区突触体钙水平的年龄差异

本实验使用荧光指示剂Ｆｕｒａ－２与Ｔｂ~（３＋）,检测了不同年龄组大鼠的不同脑区（海马、皮层、间脑、小脑）突触体内游离钙与膜结合钙水平。结果显示,与青年对照组相比,老年大鼠大部分脑区（海马、皮层、间脑）突触体内游离钙水平显著增高,尤其是海马突触体内游离钙增高极为显著;其突触体膜结合钙水平表现为：海马、小脑两脑区明显升高,而皮层、间脑两脑区明显下降,呈现一种全脑范围内的钙水平失衡。提示动物的衰老与其脑内钙自体平衡失调有关。     

An improved Eschehchia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7·9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10⁵ cfu μg^-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus , it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.     

Poly(ADP-Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damage

Abstract: DNA damage activates a nuclear enzyme poly(ADP-ribose) synthetase (PARS) that facilitates DNA repair by adding multiple ADP-ribose groups to nuclear proteins such as histones and PARS itself. N -Methyl- d -aspartate neurotoxicity may involve DNA damage excessively activating PARS to deplete its substrate NAD, as PARS inhibitors prevent this toxicity. We now show that PARS is rapidly and markedly activated in PC12 cells following treatment with neurotoxic agents, including the amyloid β-protein, hydrogen peroxide, N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its active metabolite N -methyl-4-phenylpyridine (MPP⁺). With MPP⁺, PARS activity is increased fivefold in 1 h and 20-fold by 3 h. By contrast, direct measurement of DNA damage by the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay shows no significant increase by 3 h and less than fourfold by 24 h. These findings indicate that PARS activity can provide a simple, sensitive, and early index of DNA damage following neurotoxic insults.     

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

外消旋薄荷醇对映选择性酶促酯化中酸酐与羧酸的比较研究

利用脂肪酶在有机溶剂中催化对映选择性酯化反应对外消旋薄荷醇进行了有效的光学拆分。对分别使用酸酐和相应的游离羧酸作酰基给体时的反应性能进行了比较。发现酸酐的反应性远高于对应的游离羧酸,但在酶的催化作用下酸酐易水解成为游离羧酸;在微水系统中使用过高浓度的酸酐会导致酶缺水而失活,同时会促进手性醇的非选择性酯化,从而降低产物的光学纯度。然而,在连续流加丙酸酐的半批式反应系统中,所有这些缺点均可有效地克服。与使用游离丙酸的批式反应系统相比,ｄｌ-薄荷醇的反应时间缩短了一半,酶的稳定性大幅度提高,而产物ｌ薄荷醇酯的光学纯度不相上下（＞９８％ｅ）。     

兔感觉神经特异蛋白的纯化及稳定性观察

以兔脊神经节及背根纤维为材料,通过制备匀浆,DEAE-Sephacel阴离子交换层析,高压液相凝胶过滤层析分离纯化了感觉神经特异蛋白29 ku,并进行了该蛋白的稳定性观察.     

腺苷酸激酶基因在大肠杆菌中的可溶性高表达

报道了鸡肌腺苷酸激酶基因的克隆和在温控启动子P_RP_L调控下在大肠杆菌中的可溶性高效表达.SDS-PAGE分析表明,鸡肌腺苷酸激酶的含量可占大肠杆菌细胞总蛋白含量的38％.利用Johnson等的干冰/乙醇-冰水浴反复冻融法,可将此重组蛋白进行富集,纯度可达85％以上.鸡肌腺苷酸激酶可与抗兔肌腺苷酸激酶单克隆抗体产生强的交叉反应.