Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase

Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant km. The beta(235 K) subunit of fodrin, but not the alpha(240 K) subunit, was phosphorylated by the kinase. Neither the alpha(240 K) subunit nor the beta(220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.     

SPE-HPLC法快速检测发酵液中紫杉醇的含量

针对红豆杉内生真菌发酵液中紫杉醇的含量测定进行探讨,以建立快速高效低耗的检测方法.采用C_(18)固相萃取柱对紫杉醇进行吸附,用不同浓度的甲醇-乙酸铵和甲醇分别作为洗脱剂对其进行洗脱,比较两者的洗脱效果,洗脱液用HPLC进行检测;色谱条件为:流动相甲醇(v):水(v):乙腈(v)=20: 45: 35,流速:0.70 ml/min,检测波长:227 nm.结果表明,浓度为80%的甲醇溶液洗脱效果较好,紫杉醇的回收率为87.6%.     

Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes

Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells. This resistance (> 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors. The extent of extraction did not depend on whether the cells were in monolayer culture or in suspension or whether choleragen was bound at 0 or 37°C. A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells. Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes. When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside G_M1, toxin binding was increased and the bound toxin was also resistant to extraction. When these cells were incubated with [³H]G_M1, up to 70% of the cell-associated G_M1 was extracted under the mild conditions. When the G_M1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of G_M1. Similar results were obtained with isolated membranes. When choleragen-receptor complexes were isolated from N18 cells labeled with [³H]galactose by immunoadsorption, only labeled G_M1 was specifically recovered. These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.     

para- and ortho-Pyridinium aldoximes in reaction with acetylthiocholine

In the oximolysis reaction para-aldoximes K027 and TMB-4 react faster with ATCh than ortho-aldoximes HI-6 and K033. The reaction rate constants at 25 degrees C were 22 M(-1) min(-1) for HI-6 and K033, 230 M(-1) min(-1) for TMB-4 and 306 M(-1) min(-1) for K027. Semi-empirical calculations showed that differences in rates do not origin from different electron density on the oxygen of the oxime group, but can be explained by the steric hindrance of the oxime group within the molecule. Thermodynamic parameters, DeltaG#, DeltaH# and DeltaS#, were also determined for oximolysis reaction.     

Phosphorylation of microtubule-associated protein 2 by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues

Microtubule-associated protein 2 (MAP 2) from the rat brain was phosphorylated by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues. The apparent Km for MAP 2 of Kinase II was 0.2 μ$\text{M}$. The maximum incorporation of phosphate into MAP 2 by the action of Kinase II was about 5 mol of phosphate per mol of MAP 2, while that by the action of cAMP-dependent protein kinase was about 3 mol of phosphate per mol of MAP 2. When microtubule-associated proteins were incubated with both Kinase II and cAMP-dependent protein kinase together, about 7 mol of phosphate were incorporated into 1 mol of MAP 2.     

Strand breaks of mammalian mitochondrial DNA induced by carcinogens.

Closed circular mitochondrial DNA in mammalian cells was degradated to the open circular form by exposure of the cells to the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline 1-oxide (4NQO). MNNG caused more strand scission of mitochondrial DNA than 4NQO at the same concentration. The action of the carcinogens on mitochondrial DNA did not parallel that with nuclear DNA which was damaged by 4NQO more markedly than by MNNG. Mitochondrial DNA damaged by carcinogens was not repaired during 4-20 h of post-treatment incubation of the cells. Incorporation of labeled thymidine into the closed circular mitochondrial DNA, decreased by the treatment of cells with carcinogens, recovered during post-treatment incubation.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Determination of tobacco-specific N-nitrosamines in urine of smokers and non-smokers

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN), N′-nitrosoanabasine (NAB) and N′-nitrosoanatabine (NAT) and are found in tobacco and tobacco smoke. TSNA are of interest for biomonitoring of tobacco-smoke exposure as they are associated with carcinogenesis. Both NNK and NNN are classified by IARC as Group 1 carcinogens. Samples of 24?h urine collections (n?=?108) were analysed from smokers and non-smokers, using a newly developed and validated LC-MS/MS method for determining total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major metabolite of NNK), and total NNN, NAB and NAT. TSNA levels in smokers’ urine were significantly higher than in non-smokers. In smokers, urinary excretion of total TSNA correlated significantly (r?>?0.5) with markers of smoking dose, such as daily cigarette consumption, salivary cotinine and urinary nicotine equivalents and increased with the ISO tar yield of cigarettes smoked. The correlation between urinary total NNN and the smoking dose was weaker (r?=?0.4–0.5). In conclusion, this new method is suitable for assessing tobacco use-related exposure to NNK, NNN, NAB and NAT.     

一种改进的异硫氰酸胍法结合硅胶膜离心吸附柱提取孕妇外周血微量胎儿RNA的方法

孕妇外周血中存在胎儿RNA为无创性产前诊断提供了基础.但血液中富含RNA酶和微量胎儿RNA的特点,对从孕妇血浆中提取胎儿RNA带来困难. 我们以ε血红蛋白基因和胎盘特异表达基因4（PLAC4）mRNA作为研究对象,用改进的异硫氰酸胍法结合硅胶膜离心吸附柱法探索孕妇外周血中胎儿微量RNA的提取方法,获得满意效果. 30例孕妇和9例非孕妇外周血样品中总RNA经凝胶电泳测定显示3条带,分别为28S, 18S和 5.8S. 其28S条带亮度为18S亮度的2倍.总RNA质量浓度（A260／A280）为1.97 g/L,光密度比值(A260-A320）/（A280- A 320）为1.86. 30例孕妇外周血样本有7例提取到ε血红蛋白基因mRNA,ε血红蛋白基因 mRNA 的最小浓度为0.537 μg/mL,最大浓度为1.79 μg/mL,ε血红蛋白基因mRNA的浓度中位数为124 μg/mL. 30例孕妇外周血样本提取到PLAC4 基因mRNA,浓度最小值为2.105×103 copies/mL,最大值为12.760×103 copies/mL,而9例非孕妇中均未提取到（P＜0.01）,浓度中位数为6.612×103 copies/mL. 因此,改进的异硫氰酸胍法与硅胶膜离心吸附柱纯化法相结合,可有效抑制RNA降解,用于提取、纯化孕妇血液中微量胎儿RNA.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Enzyme-bound early product of purified poly(ADP-ribose) polymerase.

Bovine thymus poly(ADP-ribose) polymerase with a purity of 99% on a SDS-poly-acrylamide gel electrophoresis was able to initiate poly(ADP-ribose) synthesis without adding any exogenous acceptor protein to the reaction system. Analyses of the early reaction product synthesized without exogenous acceptor protein revealed that the product was oligo(ADP-ribose) with a mean chain length of 2.6 and was bound tightly to the enzyme protein. When the radioactive early reaction product was chased by incubating further with cold NAD⁺, ADP-ribose unit was found to be added to the terminal AMP-residue of the oligo(ADP-ribose) attached to the enzyme. The stability of the early reaction product in high concentration of salt, strong acid, sodium dodecyl sulfate, and urea strongly suggests a covalent nature of the binding of oligo(ADP-ribose) to the enzyme.     

Inactivation and reactivation of mitochondrial respiration by charged detergents

Respiration of submitochondrial preparations can be inhibited by the cationic detergent cetyl trimethyl ammonium bromide and the anionic detergent sodium dodecyl sulfate in the range of 0.3–2 μmol of detergent per mg of mitochondrial membrane protein depending on the substrate and detergent used. This inhibition can be rapidly reversed by neutralizing a given detergent by the detergent of the opposite charge. At higher levels of the inhibiting detergent, no such reactivation was observed. Spin labeling assays of membrane structure were used to correlate structural effects with the loss and recovery of respiratory functions.Because the detergents progressively disrupt membrane structure, mitochondria were cross-linked with bifunctional imidoesters to an extent that osmotic properties and detergent lysis were gone, but respiration remained. Such fixed respiring mitochondria also show inhibition reactivation phenomena.     

Determination of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione in plasma by direct injection into a coupled column liquid chromatographic system

The chemical substance 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is in clinical use for the treatment of hereditary tyrosinemia type 1. In the present study, the plasma concentration of NTBC was determined by a coupled column liquid chromatographic method. A 20-μl volume of plasma was diluted with phosphate buffer, pH 2, and injected into a small precolumn (BioTrapAcid C₁₈) with a mobile phase containing sulfuric acid. The precolumn was based on the restricted access principle, i.e., retention of NTBC within the lipophilic pores, while polar and large endogenous compounds were eluted with the void volume. NTBC was transferred to the analytical column using a mobile phase with a high content of acetonitrile. The compound was monitored by UV detection at 278 nm. The standard curve was linear between 0.3 and 69 μM, and the between-day precision (RSD) was 3% (n=6 days) at 13.8 μM and 14% (n=6 days) at 0.3 μM NTBC in plasma. The quantitation limit was approximately 0.3 μM using 20 μl of plasma.