基因定点诱变删除及天然α型心钠素的分泌型表达

用基因定点诱变技术,删除了pO_1α ANF表达质粒中的33对碱基,使人α型心钠素结构基因直接与大肠杆菌分泌型表达质粒pIN-Ⅲ-OmPA中的信号肽酶切位点编码区相连,构成天然人α型心钠素的表达质粒pANF,在IPTG诱导下表达28肽的天然人α型心钠素。纯化后的表达产物具有天然心钠素的放免活性和很强的舒张血管的生物活性。     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Improvement of Longevity and Viability of Sperm Cells Isolated from Pollen of Zea mays L

Our previous studies showed that the common maize (Zea mays L.) sperm isolation medium (Brewbaker and Kwack salts in 0.44 m sucrose without buffering) caused cell lysis in vitro. In an attempt to remedy this situation, 6 sugars, 10 buffers, 5 pH values, and 3 membrane protective agents were screened to improve longevity and viability of isolated Zea mays sperm cells as estimated by hemacytometry and flow cytometry. Use of 0.55 m galactose in the isolation solution increased sperm yield by 2.5-fold compared with sucrose, and suspension of isolated sperm cells in the galactose solution gave the best longevity among the six sugars. Buffering the galactose solution with 2 mm 2-(N-morpholino)ethanesulfonic acid significantly improved longevity, whereas other buffers had no effect or decreased the longevity and/or viability. Among the five pH values tested (5.0, 6.0, 6.7, 7.0, and 8.0), pH 6.7 appeared to be optimal for maintenance of both longevity and viability. Screening of membrane protectants showed that cysteine caused a rapid decrease in cell viability and increased lysis, whereas dithiothreitol increased the cell numbers but lowered their viability. Addition of 0.1% bovine serum albumin increased cell numbers and viability, and about 70% of the cells remained viable after 72 h of suspension. Cell longevity and viability were also improved in 0.44 m sucrose when the solution was conditioned with 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin. Use of 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin inthe isolation and suspension medium significantly improved the viability and longevity of sperm cells isolated from Zea mays pollen.     

Active site conformation in myoglobin as determined by X-ray absorption spectroscopy

X-ray absorption fine structure experiments were performed to study structural and dynamic aspects of the active site of various forms of myoglobin. The structures determined for deoxyMb, MbCO, and MbO2 are consistent with the structure established by X-ray absorption fine structure experiment and X-ray crystallography. The first shell of ferrous MbNO determined contains 5 nitrogens located at 2.02 A and a short NO bond length of 1.76 A. This study focuses on the change of the XAFS Debye-Waller factor with temperature, which is a measure of thermal and static disorder. It was found that the changes of Debye-Waller factor with temperature for the Mb proteins, except deoxyMb, are consistent with a simple Einstein model, in which a single frequency was assumed for the bond stretching modes. In contrast, the temperature dependence of deoxyMb cannot be fitted to the Einstein model and a large disorder was found at low temperatures, which indicates the existence of conformational substates of the active site.     

Determination of trimethylamine and related aliphatic amines in human urine by head-space gas chromatography

A rapid and simple assay procedure employing head-space gas chromatography has been developed for the routine quantification of volatile methylamines and stable trimethylamine N-oxide present in human urine. This assay will enable the rapid screening of patients and aid the diagnosis of fish odour syndrome.     

Genetic and Molecular Organization of Ribosomal DNA (Rdna) Variants in Wild and Cultivated Barley

Twenty rDNA spacer-length variants (slvs) have been identified in barley. These slvs form a ladder in which each variant (with one exception) differs from its immediate neighbors by a 115-bp subrepeat. The 20 slvs are organized in two families, one forming an eight-step ladder (slvs 100-107) in the nucleolus organizer region (NOR) of chromosome 7 and the other a 12-step ladder (slvs 108a-118) in the NOR of chromosome 6. The eight shorter slvs (100-107) segregate and serve as markers of eight alleles of Mendelian locus Rrn2 and the 12 longer slvs (108a-118) segregate and serve as markers of 12 alleles of Mendelian locus Rrn1. Most barley plants (90%) are homozygous for two alleles, including one from each the 100-107 and the 108a-118 series. Two types of departures from this typical pattern of molecular and genetic organization were identified, one featuring compound alleles marked by two slvs of Rrn1 or of Rrn2, and the other featuring presence in Rrn1 of alleles normally found in Rrn2, and vice versa. The individual and joint effects on adaptedness of the rDNA alleles are discussed. It was concluded that selection acting on specific genotypes plays a major role in molding the strikingly different allelic and genotypic frequency distributions seen in populations of wild and cultivated barley from different ecogeographical regions.     

Post-translational modification of a monocyte-specific chemoattractant synthesized by glioma, osteosarcoma, and vascular smooth muscle cells

Chemotaxis is an important step in monocyte recruitment in inflammation, wound healing, and tumor growth. We reported previously that monocyte chemotactic activity secreted by malignant cells and normal smooth muscle cells is associated with a protein or family of proteins that are related to the monocyte-specific smooth muscle cell-derived chemotactic factor (SMC-CF) (Graves, D. T., Jiang, Y. L., Williamson, M. J., and Valente, A. J. (1989) Science 245, 1490-1493). Similar monocyte chemotactic proteins (MCP-1) produced by U-105MG human glioma cells have also been identified (Yoshimura, T., Robinson, E. A., Tanaka, S., Appella, E., Kuratsu, J., and Leonard, E. J. (1989) J. Exp. Med. 169, 1449-1459). We now report that the MCP-1 gene is expressed in MG-63 human osteosarcoma and vascular smooth muscle cells and that SMC-CF antiserum specifically immunoprecipitates proteins synthesized by U-105MG glioma cells. Experiments were undertaken to elucidate the processing pathway of MCP-1/SMC-CF-like proteins in each of these cell types. These experiments demonstrate that larger MCP-1/SMC-CF-like proteins are derived from a Mr = 9000 precursor. Post-translational modification involves the addition of O-linked carbohydrates and sialic acid residues. Differences in carbohydrate processing account for the heterogeneity in MCP-1/SMC-CF-like proteins produced by different cell types. Secretion of these proteins occurs rapidly following processing events in the endoplasmic reticulum-Golgi compartment.