Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates

Oligodeoxyribonucleoside methylphosphonates of defined sequence of the type d-Np(NP)nN, where n is 6-13, are readily prepared on insoluble polystyrene supports by use of protected 5'-(dimethoxytrityl)deoxyribonucleoside 3'-(methylphosphonic imidazolides) as synthetic intermediates. The imidazolides are prepared in situ by reaction of protected 5'-(dimethoxytrityl)deoxyribonucleoside with methylphosphonic bis(imidazolide) and can be stores in the reaction solution for up to 2 weeks at 4 degrees C with no loss in activity. The condensation reaction is accelerated by the presence of tetrazole, which appears to act as an acid catalyst. The half-life for dimer formation on the polystyrene support is 5 min, and the reaction is 95% complete after 60 min. Although similar kinetics are observed when controlled pore glass is used as the support, the extent of the reaction does not go beyond 78%, even after prolonged incubation. In order to simplify purification and sequence analysis of the oligomer, the 5'-terminal nucleoside unit is linked via a phosphodiester bond. This linkage may be introduced by either an o-chlorophenyl phosphotriester method or a cyanoethyl phosphoramidite method. The latter procedure simplifies the deprotection step, since the cyanoethyl group is readily cleaved by ethylenediamine, which also removes the base protecting groups and cleaves the oligomer from the support. The singly charged oligomers are easily purified by affinity chromatography on DEAE-cellulose. The chain lengths of the oligomers were confirmed after 5'-end labeling with polynucleotide kinase by partial hydrolysis of the methylphosphonate linkages with 1 M aqueous piperidine followed by polyacrylamide gel electrophoresis of the hydrolysate.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo or in vitro selection for resistance to natural cytotoxic cell lysis selects for variants with increased tumorigenicity

Experiments were designed to test the hypothesis that transformed cells that are NC sensitive must escape NC activity if they are to grow as tumors in normal individuals. NC-resistant variants were selected either in vivo or in vitro from NC-sensitive cell lines that grow as tumors in immunodeficient mice but not in syngeneic normal mice. The tumorigenicity of cloned NC-resistant variants was compared with the parental cell lines and to cell lines that went through the selection procedure, but after cloning remained NC sensitive. Cloned NC-resistant cell lines derived from tumors that developed in x-irradiated nude mice after the injection of an NC-sensitive cell line are tumorigenic in normal mice, whereas cloned NC-sensitive cell lines derived from the same tumors are unable to grow as tumors in normal mice. Similarly, six of seven NC-resistant cloned cell lines independently isolated after in vitro selection for NC-resistance are tumorigenic in normal mice, whereas cloned NC-sensitive cell lines isolated from the same in vitro selected populations are not tumorigenic in normal mice. Thus, either the in vivo or in vitro selection of NC-resistant cells selects for cells tumorigenic in normal mice; these findings, along with our previous observations that selection for cells tumorigenic in normal mice selects for NC resistance, provide compelling evidence that escape from NC activity is required before some transformed cells can grow as tumors in normal mice.     

Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase

Fumarate reductase (encoded by frd) and succinate dehydrogenase (encoded by sdh) of Escherichia coli are both known to catalyze the interconversion of fumarate and succinate. Fumarate reductase, however, is not inducible aerobically and therefore cannot participate in the dehydrogenation of succinate. Three classes of suppressor mutants, classified as frd oxygen-resistant [frd(Oxr)], constitutive [frd(Con)], and gene amplification [frd(Amp)] mutants, were selected from an sdh strain as pseudorevertants that regained the partial ability to grow aerobically on succinate. All contained increased aerobic levels of fumarate reductase activity. In frd(Oxr) mutants expression of the operon showed increased resistance to aerobic repression. Under anaerobic conditions expression of the operon became less dependent on the fnr+ gene product, a pleiotropic activator protein for genes encoding anaerobic respiratory enzymes. Exogenous fumarate, however, was still required for full induction, and repression by nitrate was undiminished. Thus, aerobic repression and anaerobic nitrate repression appear to involve separate mechanisms. In frd(Con) mutants expression of the operon became highly resistant to aerobic repression. Under anaerobic conditions expression of the operon no longer required the fnr+ gene product or exogenous fumarate and became immune to nitrate repression. In partial diploids bearing an frd(Oxr) or an frd(Con) allele and phi(frd+-lac) there was no mutual regulatory influence between the two genetic loci. Thus, the frd mutations act in cis and hence are probably in the promoter region. In frd(Amp) mutants the frd locus was amplified without significant alteration in the pattern of regulation.     

Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments

We describe herein the purification of a protein from skeletal muscle that binds to ("caps") the morphologically defined barbed end of actin filaments. This actin-capping protein appeared to be a heterodimer with chemically and immunologically distinct subunits of Mr = 36,000 (alpha) and 32,000 (beta), Rs = 37 A, s20,w = 4.0 S, and a calculated native molecular weight of approximately 61,000. The protein was obtained in milligram quantities at greater than 95% purity from acetone powder of chicken skeletal muscle by extraction in 0.6 M KI, precipitation with ammonium sulfate, sequential chromatographic steps on DEAE-cellulose, hydroxylapatite, and Sephacryl S-200, followed by preparative rate zonal sucrose density gradient centrifugation. In immunoblots of myofibrillar proteins, affinity-purified antibodies selectively recognized protein bands of the same molecular weight as the subunits of the capping protein to which they were made, indicating that the isolated capping protein is a native myofibrillar protein, and not a proteolytic digestion product of a larger muscle protein. A specific interaction of the capping protein with the barbed end of actin filaments was indicated by its ability to inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and inhibit the addition of actin monomers to the barbed end of heavy meromyosin-decorated actin filaments as determined by electron microscopy. All of these effects occurred at nanomolar concentrations of capping protein and micromolar concentrations of actin, suggesting a high affinity interaction.     

A re-examination of the interaction of vinculin with actin

Vinculin prepared by published procedures (i.e., Feramisco, J. R., and K. Burridge, 1980, J. Biol. Chem., 255:1194-1199) contains contaminants that have been shown by Evans et al. (Evans, R. R., R. M. Robson, and M. H. Stromer, 1984, J. Biol. Chem., 259:3916-3924) to reduce the low-shear viscosity of F-actin solutions. In this study we separated contaminants from conventional vinculin preparations by hydroxylapatite chromatography. We found that although the contaminants represented a small fraction (less than or equal to 5%) of the total protein in the conventional vinculin preparations, they were responsible for practically all of the filament capping and bundling activities previously attributed to vinculin. In addition, we examined the size of the molecule(s) responsible for the observed capping activity and found that its apparent molecular weight under denaturing conditions in sodium dodecyl sulfate (SDS) polyacrylamide gels fell within a broad range of 23,000-33,000. These results contrast with the observation that under nondenaturing conditions, the activity migrated in gel filtration columns at a position that corresponded to the Stoke's radius of a much bigger molecule. Since the migration of the activity in these chromatographic experiments is independent of the presence of vinculin, it is unlikely that the active protein associates with vinculin with high affinity under the conditions examined.     

Isolation and characterization of a 37,000-dalton protein associated with the erythrocyte membrane

We have purified a 37,000-dalton polypeptide (p37) from the red cell membrane that was found in previous studies to undergo a lineage-specific alteration in its membrane association. Our data suggest that p37 associates with the red cell membrane through electrostatic interactions that are resistant to 0.5 M NaCl or 10 mM EDTA. Conditions found to elute p37 from red cell ghosts include H2O at pH 12, 0.1 N NaOH + 1 mM ethanol and 1.0% Triton X-100. p37 was purified substantially from ghosts by Triton X-100 solubilization followed by sequential DEAE-Sephadex and CM-Sephadex chromatography. When p37 was analyzed by two-dimensional gel electrophoresis, a family of isoelectric focusing variants was detected ranging in pI from 7.0 to 7.8. All of the isoelectric focusing variants showed homology to one another when compared serologically with anti-p37 antibodies or by limited peptide mapping using Staphylococcus aureus V8 protease. The isoelectric focusing variants appear to represent distinct, yet related polypeptides rather than degrees of post-translational modifications to a single species, inasmuch as all of the variants are present in anti-p37 immunoprecipitates prepared from in vitro translations programmed with p37 mRNA.     

北京东郊作物土壤系统中重金属的迁移、分布、积累

 本文研究了北京东郊污灌区重金属在作物—土壤中的迁移、分布、积累规律,证实本区蔬菜中汞含量比粮食作物约大3—15倍,比水果约大6—200倍。麦粒、糙米中的Cu、Hg、Cd、Pb、Ni的含量与土壤含量相关性不显著。架豆中重金属含量与土壤中重金属含量的相关性,只有Zn,Pb达显著水平。白菜土有机质含量与重金属含量相关性达显著水平,而白菜的重金属含量与土壤的重金属含量相关性却不显著。说明除了土壤中重金属的总量外,有效态含量的多少,是影响本区作物吸收积累重金属的主要因素。 本区施污泥的土壤和生长的作物Cd／Zn大部小于1%、盆栽试验证明：施用本区污泥污水对水稻生长发育的影响比施污泥灌清水的影响大些,因此,施用含重金属污泥时,最好不要超过5000斤／亩。大田和室内模拟试验证明：重金属从土壤中迁移到植物,由植物带走输出的量极少,其中以带走输出的Hg、Cd,As相对较多,带走输出的Pb、Cr相对的少些。     

呼吸链底物和抑制剂对线粒体内膜流动性的影响

用DPH和ANS标记大鼠肝线粒体内膜,以稳态荧光偏振法,研究了呼吸链底物和抑制剂对内膜流动性的影响。1.苹果酸+谷氨酸、琥珀酸分别为底物,均能引起内膜流动性增加。2.琥珀酸对含心磷脂的脂质体的膜流动性无影响。3.在鱼藤酮存在的条件下,苹果酸+谷氨酸对内膜流动性的增加作用消失,但琥珀酸的作用仍然存在。有氰化钾时则琥珀酸的作用消失。4.不论外加底物存在与否,鱼藤酮使内膜的流动性下降,而氰化钾则使之增加。抗霉素A亦可使内膜的流动性增加。上述结果表明:线粒体内膜流动性与其功能密切相关。电子沿呼吸链传递使线粒体内膜流动性增加,这种变化可能与呼吸链成分的氧化还原态有关。     

我国东喜马拉雅及其邻区牛肝菌目的研究（续）

本属与多种树种有菌根关系：如Pinus,Larix,Abies和Pseudotsuga,但在Larix林下,本菌往往与土壤中的鞣料相聚集,对周围某些植物根系不利。本属现知15种,本区6种。