Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

磷脂模型膜的时间分辨纳秒荧光光谱的研究

叙述了用时间分辨率纳秒荧光技术研究大豆磷脂或合成磷脂模型膜（脂质体）的物理状态变化与荧光寿命以及时间分辨各向异性测量参数变化相关性的实验结果。用荧光探剂ＭＣ５４０标记模型膜的结果表明,荧光寿命（τ）的变化与脂质／探剂比例、磷脂组成、磷脂的极性头部、脂肪酸酰链长度等有关。用ＤＰＨ（１,６－ｄｉｐｈｅｎｙｌ－１,３,５－ｈｅｘａｔｒｉｅｎｅ）标记磷脂模型膜的时间分辨各向异性分析结果表明,序参数（Ｓ）、     

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.     

Conformational analysis of a snake neurotoxin by prediction from sequence, circular dichroism, and raman spectroscopy.

The secondary structure of the major neurotoxin from the sea snake Lapemis hardwickii was investigated by several methods of conformational analysis: structure prediction, circular dichroism, and laser Raman spectroscopy. From the primary structure, secondary structure prediction yielded two regions of β-sheet structure at residues 1–7 and 41–45. β-Turns were predicted at residues 14–17, 20–23, 30–33, 37–40, and 46–49. From the predictions, the toxin appears to be composed of approximately 20% β-sheet and 33% β-turn. The CD spectrum of the native toxin appears to be a hybrid of model spectra for β-sheet and β-turn proteins. The pH perturbation studies on the toxin observed by CD demonstrated that the toxin is a very stable molecule except at extremely high or low pH values. The Raman data indicated that the toxin contains both antiparallel β-sheet and β-turn structure. Using two methods of secondary structure quantitation from Raman spectra the molecule was calculated to contain 35% β-sheet from one method and 27% from the other. Overall, the various methods demonstrate that the toxin is composed of β-sheet and β-turn structure with little or no α-helix present. From the comparison of these different techniques appreciation can be gained for the necessity of several methods when identifying and quantitating secondary structure.     

Purification of chicken brain choline acetyltransferase

Chicken brain choline acetyltransferase was purified to homogeneity using ammonium sulfate fractionation, followed by chromatography on DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150, immunoabsorption and Sepharose-CoA columns. A purification of 3500-fold was achieved and the final preparation had a specific activity of 2:32 μmol acetylcholine formed per minute per milligram protein. The purified chicken choline acetyltransferase migrated as a single band on polyacrylamide gel electrophoresis in the presence and absence of sodium deodecyl sulfate. The native enzyme, with a molecular weight of 67,000 daltons, consists of two subunits of identical molecular weight. Chicken choline acetyltransferase has a sharp pH optimum of 7.4. It is activated by sodium chloride and potassium chloride but inhibited by cupric ion and N-ethylmaleimide.     

两株粗糙型沙门氏菌对小鼠抗异源G~-杆菌攻击的免疫保护研究

本文对引自日本的一株粗糙化学型变异株明尼苏达沙门氏菌Re595(J)和引自美国的一株Re595(A)对小鼠异源性G~-杆菌主动和被动保护作用进行了比较。结果表明,两株菌对异源G~-杆菌的大肠杆菌和变形杆菌攻击均有良好的保护作用,但Re595(J)对抗肺炎克雷伯氏杆菌攻击的保护作用明显优于Re595(A),而Re595(A)抗绿脓杆菌攻击的保护作用则明显优于Ke595(J)。表明两株Re595的免疫原存在着差异。     

菌种与品种最佳组合的选育及增产效果

本文通过多年多点的田间接种试验,分析了大豆根瘤菌C_(33)(系VSDA_(110)的突变株)的增产效应.在合丰25号大豆接种C_(33)取得明显的增产效果,两年平均比CK增产28.55%,比61A76增产24.5%;C_(33)接种在其他品种以及在不同类型和肥力的土壤上增产效果也均高于CK和61A76,说明大豆根瘤菌C_(33)比当前生产上应用的61A76更具有广谱性和高效性.     

Fulvic acid disturbs processing of procollagen II in articular cartilage of embryonic chicken and may also cause Kashin-Beck disease.

Kashin-Beck disease is an endemic osteoarthropathy in China which may lead to skeletal deformation and dwarfism. We have analysed articular cartilage from two patients and found an accumulation of the precursor molecule, pro-pN-collagen II (pN, peptide attached at the amino-terminus) which was not present in extracts of control fetal cartilage. In addition, collagen II isolated from the same tissue by limited pepsin digestion had a decreased electrophoretic mobility, increased proline hydroxylation and decreased thermal stability. Previously, a genetic defect in pro-pN-collagen-I processing has been described in calf and sheep (dermatosparaxis) and man (Ehlers-Danlos, type VII) which caused an extreme fragility of the skin [Lenaers, A., Ansay, M., Nusgens, B.V. & Lapière, C.M. (1971) Eur. J. Biochem. 23, 533-541; Helle, O. & Nes, N.J. (1972) Acta Vet. Scand. 13, 443-445; Lichtenstein, J.R., Martin, G.R., Kohn, L.D., Byers, P.H. & McKusick, V.A. (1973) Science 182, 298-300]. Accordingly, one may assume that the impaired conversion of pro-pN-collagen II to collagen II and the structural alteration of collagen II, presumably caused by fulvic acid and other environmental factors, play an important role in the pathogenesis of Kashin-Beck disease.