Effect of Carbachol and 56 mM-Potassium Chloride on the Cyclic AMP-Mediated Induction of Tyrosine Hydroxylase in Neuroblastoma Cells in Culture

Abstract: Tyrosine hydroxylase (TH) activity is increased two- to threefold in neuroblastoma cell line NBP² maintained in culture for 48 h in the presence of either the inhibitor of cyclic AMP-phosphodiesterase (PDE), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20- 1724), or the activator of adenylate cyclase, prostaglandin E₁ (PGE₁). Cyclic AMP levels are elevated 70–80% and 30–40% throughout the 48-h treatment with RO 20-1724 and PGE₁, respectively. Carbachol does not affect either basal TH activity or cyclic AMP levels in the cells. However, the cholinergic agonist delays the induction of TH elicited by either RO 20-1724 or PGE₁. This delay is prevented by atropine. The elevation in cyclic AMP levels elicited by either RO 20-1724 or PGE₁ is blocked for 1 h or 15 min. respectively, after treatment with carbachol. Cyclic AMP levels then begin to rise until they reach those levels observed in the presence of RO 20-1724 or PGE₁ alone by 12 h or 1 h of treatment, respectively. Time course studies demonstrate that this transient inhibition of the elevation of cyclic AMP is associated with a 48-h delay in the induction of TH elicited by either RO 20-1724 or PGE₁. In contrast, the induction elicited by 8-bromo cyclic AMP is unaffected by carbachol. A depolarizing concentration (56 mM) of KCl produces a 24-h delay in the induction of TH elicited by RO 20-1724, without affecting the concomitant elevation of cyclic AMP produced by the PDE inhibitor. Furthermore, 56 mM-KCl inhibits the induction of TH elicited by 8-bromo cyclic AMP. It thus appears that carbachol delays the induction of TH by transiently inhibiting the elevation of cyclic AMP, whereas potassium depolarization delays the induction of TH by inhibiting a process with a site of action that is distal to the elevation of cyclic AMP.     

Induction of vancomycin resistance in Enterococcus faecium by non-glycopeptide antibiotics

Abstract Bacitracin and other antibiotics that inhibit late stages in peptidoglycan biosynthesis induce vancomycin resistance in a high-level, inducibly vancomycin-resistant strain of Enterococcus faecium . Exposure to bacitracin led to synthesis of the lactate-containing UDP-MurNAc-pentadepsipeptide precursor required for vancomycin resistance. These findings indicate that inhibition of peptidoglycan biosynthesis can lead to induction of vancomycin resistance and raise the possibility that multiple signals may serve to induce resistance.     

Bleomycin-induced β-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues

Abstract A bleomycin-resistance gene, designated blmA , has been cloned from bleomycin-producing Streptomyces verticillus by Sugiyama et al. (Gene 151 (1994) 11–16). The present study shows that Escherichia coli harboring the blmA -carrying pUC plasmid overproduced β-lactamase, encoded by an ampicillin-resistance gene on the plasmid, when cultured in the presence of bleomycin, which suggests that bleomycin may act as an inducer (or an activator) for the expression of the specific gene in the presence of blmA . We constructed a vector, designated pMAB50, which senses bleomycin and produces a pigment, using blmA and a Streptomyces tyrosinase gene located under the control of β-lactamase promoter: E. coli harboring pMAB50 produced the melanin pigment in the presence of bleomycin-type antibiotics, suggesting that the transformed E. coli can be employed as a reporter organism to screen bleomycin analogues.     

Multiple forms of O-methyltransferase involved in the microbial conversion of abietic acid into methyl abietate by Mycobacterium sp.

Six out of seven tested strains of mycobacteria transformed abietic acid to methyl abietate in shake culture. The conversion carried out by Mycobacterium sp. MB 3683 was induced by the substrate and stimulated by methionine. Fractionation of the cell extract of Mycobacterium sp. MB 3683 on DEAE cellulose, Ultrogel AcA 44 and MONO Q resulted in the separation of three distinct methyltransferase activities which could also esterify palmitic acid. The separated forms of the methyltransferase exhibited different activities towards these two substrates.     

重组人VEGF165的表达纯化及单抗的初步筛选

血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)是抑制肿瘤生长和转移的重要靶点。为获得抗VEGF单抗细胞株,构建了rhVEGF₁₆₅工程菌,并利用复合自动诱导获得高效表达。经纯化获得高纯度rhVEGF₁₆₅蛋白,经检测具有促人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)增殖活性,其EC₅₀为2.4ng/ml。免疫小鼠,获得了3株能稳定分泌抗VEGF单抗的杂交瘤细胞株,为开发VEGF治疗性单抗提供了重要基础。     

氟喹诺酮类药物体外诱导肺炎克雷伯菌耐药后GyrA和ParC的变异

目的了解3种氟喹诺酮类（FQS）体外诱导肺炎克雷伯菌（Klebsiella pneumoniae,Kpn）耐药性的差异,研究肺炎克雷伯菌DNA旋转酶A亚单位（GyrA）和拓扑异构酶ⅣC亚基（ParC）的变异与其耐喹诺酮类药物的关系。方法采用环丙沙星（CW）、左氧氟沙星（LEX）和加替沙星（GAT）对从临床分离8株Kpn进行体外分步诱导,采用琼脂平板二倍稀释法测定CIP、LVF及GAT对Kpn诱导前、后的最低抑菌浓度（MIC）,并对诱导成功的17株Kpn的GyrA的基因（gyrA）和ParC的基因（parC）进行PCR扩增,选取其中8株KpnDNA测序并进行序列分析比较。结果8株耐FQS菌株都存在GyrA变异,同FQS耐药性相关的变异有Ser83（TCC）→Phe（TTC）、Ile（ATC）和Tyr（TAC）,Asp87（GAC）→Ala（GCC）、ma（GCC）和Glu（GAA）,5株Kpn同时存在ParC的变异：丝氨酸Ser80（AGC）→Ile（ATC）。结论本研究体外实验证实了Kpn可在长期低剂量的接触抗菌药物后形成耐药菌株。在高度耐FQS的Kpn中同时存在GyrA和ParC变异。     

不同应激因子对小鼠肝脏金属硫蛋白诱导合成的影响

目的筛选出小鼠肝脏金属硫蛋白（MT）合成量最大的诱导方式。方法从时间-效应和剂量-效应两方面研究了重金属元素（Cd）、微量元素（Zn）、重金属与微量元素的组合（Cd＋Zn）、生理因子（饥饿）及创伤因子等五大类组合应激因子、19种诱导方式对小鼠肝脏中MT诱导合成的影响及效果。结果生理因子诱导MT量最小,饥饿诱导小鼠肝脏MT的量随饥饿程度的加重而增加,但各组间差异不显著（P〉0.05）;创伤因子诱导产生MT的量最高,其诱导量随创伤恢复时间的增加而降低,各组之间差异显著（P〈0.01）,本实验诱导峰值（9.0241±0.6441μmol/g）出现在创伤后6 h;重金属元素和微量元素诱导量居中,且两者混合诱导量比单独诱导量之和要大。结论成功筛选出诱导小鼠肝脏MT合成最有效的因子和最佳时间,为进一步大量合成MT及研究其功能等奠定基础。     

The discovery of state transitions in photosynthesis 40 years ago

In the late 1960s, I identified an aspect of the kinetics of chlorophyll fluorescence in algal cells that I was unable to explain in terms of photochemical quenching. I proposed a novel regulatory mechanism for the distribution of light energy to photosystems I and II, which is now known by the term of “state transitions.” I also examined the “cation-dependent redistribution of light energy” to photosystems I and II and the “energy-dependent quenching” of chlorophyll fluorescence. At that time, financial constraints prevented me from measuring the emission and action spectra of chlorophyll fluorescence at liquid-nitrogen temperature and the light quality-dependent changes in the yield of chlorophyll fluorescence at room temperature. The financial problems were solved by constructing several pieces of electronic equipment using skills obtained by repairing radios when I was a high-school and college student.