人肝癌细胞表皮生长因子受体以及佛波酯对它的调度

Using radioligand binding assay, the presence of epidermal growth factor (EGF) receptors in cells of two human liver cancer cell lines, BEL-7402 and SMMC-7721, was demonstrated. The ligand binding data were analyzed by a computer program. The dissociation constants (KD) of the ligand-receptor binding complex at equilibrium for 7402 and 7721 cells were 1.2 nM and 0.8 nM respectively, and their number of EGF receptors per cell were 6.2 x 10(4) and 2.5 x 10(4) respectively. After the treatment of cells with phorbol 12-myristate 13-acetate (PMA), no change either in the affinity or in the number of EGF receptors was found in 7721 cells. However, in the case of 7402 cells, while the number of receptors, like 7721 cells, remained unchanged, the affinity of EGF receptors displayed a time dependent modulation after PMA treatment. It dropped within the first hour to a KD value of 3.0 nM and then gradually returned to the normal control value at 48 hours or even slightly higher than normal (0.95 nM) at 96 hours of treatment. The modulation or down-regulation of EGF receptors by PMA in 7402 cells was paralleled by the simultaneous inhibition of DNA synthesis in these cells as evidenced from their reduction of 3H-TdR uptake. It is not clear what is the basis for the differences found between 7402 cells and 7721 cells in their number of EGF receptors per cell and their responsiveness to PMA treatment. It might be related to their difference in autocrine secretion of alpha-transforming growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

One-electron reduction of chromate by NADPH-dependent glutathione reductase

Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(VI) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl (.OH) radical formation during the GSSG-R catalyzed reduction of Cr(VI) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the .OH radical and Cr(V) formation, indicating that the O2- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the .OH radical formation, indicating the involvement of hydrogen peroxide (H2O2) in the reaction mechanism. Addition of H2O2 suppresses Cr(V) and enhances the .OH radical formation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(VI) by GSSG-R, react with H2O2 to generate .OH radicals, which might participate in the initiation of Cr(VI) carcinogenicity.     

Ⅱ型限制性核酸内切酶NcrⅠ的研究

 在肉色诺卡氏菌C-212株Nocardia carnea C-212中筛选到一种Ⅱ型限制性核酸内切酶NcrⅠ,经与BglⅡ的λDNA降解物的酶谱比较,以及酶识别特异性和切割位点的检测,证明了NcrⅠ是已知的限制酶BglⅡ的同切限制酶,而且其切割位点也与BglⅡ相同,其为:     

Fibronectin,not laminin,mediates heparin-dependent heparin-binding growth factor type I binding to substrata and stimulation of endothelial cell growth

Summary Fibronectin and heparin-binding growth factors (HBGF) are essential for growth of cultured endothelial cells. The stimulation of endothelial cell growth by HBGF type one (HBGF-1) in particular requires heparin or a similar glycosaminoglycan. The requirement for fibronectin and heparin for HBGF-1-stimulated endothelial cell growth may be related. HBGF-1 absorbed to the natural subcellular matrix of endothelial cells supports cell growth. [¹²⁵I]HBGF-1 specifically associates with a sequentially reconstituted matrix of collagen-fibronectin-heparin, and HBGF-1 absorbed to the reconstituted matrix supports growth of the endothelial cells. A reconstituted matrix of collagen-laminin-heparin neither supported binding of [¹²⁵I]HBGF-1 nor HBGF-1-stimulated endothelial cell growth. Association kinetics of [¹²⁵I]HBGF-1 to heparinlike sites and membrane receptor sites on endothelial cell monolayers suggest that fibronectin-heparinlike binding sites in the subcellular matrix may be an obligatory reservoir of active HBGF-1 that binds to specific cell membrane receptors. This work was carried out in the laboratory of Dr. W. L. McKeehan and supported in part by grants CA37589, DK35310 and DK38639 from the Public Health Service, Department of Health and Human Services, Washington, DC.     

Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA.

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

用光谱技术鉴定植物色素突变体

1983年我国报道了从γ-射线处理的“矮杆齐”大麦中得到了一株黄绿色的突变体1832C[1]。本文用光谱技术对该突变体的光合色素成分进行了鉴定。1　材料和方法　　材料为六棱裸大麦“矮杆齐”和由该品种大麦诱变形成的黄绿色突变体1832C(Mb1832C),以及作为对照的缺失Chlb的突变体大麦Chlorina-f2[2]都于3月初播种于实验田中。　　每个样品取30g新鲜的叶片,先用自来水后用蒸馏水冲洗干净。把洗净的叶片摊放在干净的纱布上吸干表面水分,剪碎,加入100mL预冷的含有0.4mol/L山梨醇、0.1mol/LTris-HCl(pH7.6)的缓冲液,用组织捣碎机先慢速捣碎1…