硫酸镁对大鼠海马CA1区神经元钠电流的抑制作用

利用全细胞膜片钳技术研究了硫酸镁 (MgSO4 )对大鼠海马CA1区神经元钠电流的影响。结果表明 ,MgSO4 可浓度依赖和电压依赖地抑制钠电流 ,半数抑制浓度为 4 0 5mmol/L。这一抑制作用与刺激频率无关。结果还表明 ,4mmol/LMgSO4 不影响钠电流的失活过程 ,却使半数激活电压由 - 5 5 8± 6 8mV变为 - 3 4 2± 6 2mV (n =8,P <0 0 1) ,而激活曲线的斜率因子不变。结果提示 ,MgSO4 抑制大鼠海马CA1区神经元的钠电流可能是其抗缺血缺氧造成的中枢神经系统损伤的机制之一     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.     

On the interaction of colicin E3 with the ribosome

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone     

Route of administration of chimeric BPV1 VLP determines the character of the induced immune responses

To examine the mucosal immune response to papillomavirus virus-like particles (PV-VLP), mice were immunized with VLP intrarectally (i.r.), intravaginally (i.va.) or intramuscularly (i.m.) without adjuvant. PV-VLP were assembled with chimeric BPV-1 L1 proteins incorporating sequence from HIV-1 gp120, either the V3 loop or a shorter peptide incorporating a known CTL epitope (HIVP18I10). Antibody specific for BPV-1 VLP and P18 peptide was detected in serum following i.m., but not i.r. or i.va. immunization. Denatured VLP induced a much reduced immune response when compared with native VLP. Immune responses following mucosal administration of VLP were generally weaker than following systemic administration. VLP specific IgA was higher in intestine washes following i.r. than i.va. immunization, and higher in vaginal washes following i.m. than i.r. or i.va. immunization. No differences in specific antibody responses were seen between animals immunized with BPV-1 P18 VLP or with BPV-1 V3 VLP. Cytotoxic T lymphocyte precursors specific for the P18 CTL epitope were recovered from the spleen following i.m., i.va. or i.r. immunization with P18 VLP, and were similarly detected in Peyer's patches following i.m. or i.r. immunization. Thus, mucosal or systemic immunization with PV VLP induces mucosal CTL responses and this may be important for vaccines for mucosal infection with human papillomaviruses and for other viruses.     

Murine EMT-6 carcinoma: high therapeutic efficacy of microbeam radiation therapy

Microbeam radiation therapy is an experimental modality using parallel arrays of thin (<100 micro m) slices of synchrotron-generated X rays (microplanar beams, microbeams). We used EMT-6 murine mammary carcinoma subcutaneously inoculated in the hind legs of mice to compare the therapeutic efficacies of single-fraction, unidirectional (1) "co-planar" microbeams (an array of vertically oriented microplanar beams), (2) "cross-planar" microbeams (two arrays of parallel microbeams propagated in the same direction, one with vertically and the other with horizontally oriented microplanar beams), and (3) seamless (broad) beams from the same synchrotron source. The microbeams were 90 micro m wide and were spaced 300 micro m on center; the median energy in all beams was 100 or 118 keV. Tumor ablation rates were 4/8, 4/8 and 6/7 for a 410-, 520- and 650-Gy in-slice cross-planar microbeam dose, respectively, and 1/8, 3/8, 3/7 and 6/8 for a 23-, 30-, 38- and 45-Gy broad-beam dose, respectively. When the data were pooled from the three highest doses (same average tumor ablations of 50-60%), the incidences of normal-tissue acute toxicity (moist desquamation and epilation) and delayed toxicity (failure of hair regrowth) were significantly lower for cross-planar microbeams than broad beams (P < 0.025). Furthermore, for the highest doses in these two groups, which also had the same tumor ablation rate (>75%), not only were the above toxicities lower for the cross-planar microbeams than for the broad beams (P < 0.02), but severe leg dysfunction was also lower (P < 0.003). These findings suggest that single-fraction microbeams can ablate tumors at high rates with relatively little normal-tissue toxicity.     

Development and applications of in-gel CNBr/tryptic digestion combined with mass spectrometry for the analysis of membrane proteins

Hydrophobic membrane proteins often have complex functions and are thus of great interest. However, their analysis presents a challenge because they are not readily soluble in polar solvents and often undergo aggregation. We present a sequential CNBr and trypsin in-gel digestion method combined with mass spectrometry for membrane protein analysis. CNBr selectively cleaves methionine residues. But due to the low number of methionines in proteins, CNBr cleavage produces a small number of large peptide fragments with MWs typically >2000, which are difficult to extract from gel pieces. To produce a larger number of smaller peptides than that obtained by using CNBr alone, we demonstrate that trypsin can be used to further digest the sample in gel. The use of n-octyl glucoside (n-OG) to enhance the digestion efficiency and peptide recovery was also studied. We demonstrate that the sensitivity of this membrane protein identification method is in the tens of picomole regime, which is compatible to the Coomassie staining gel-spot visualization method, and is more sensitive than other techniques reported in the literature. This CNBr/trypsin in-gel digestion method is also found to be very reproducible and has been successfully applied for the analysis of complex protein mixtures extracted from biological samples. The results are presented from a study of the analysis of bacteriorhodopsin, nitrate reductase 1 gamma chain, and a complex protein mixture extracted from the endoplasmic recticulum membrane of mouse liver.     

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500pm (45%) and 5nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.     

白鲜皮中白鲜碱含量测定

白鲜皮中白鲜碱含量测定朱丹妮徐强邱南生（中国药科大学,南京２１００３８）ＴｈｅｑｕａｎｔｉｔａｔｉｖｅｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｄｉｃｔａｍｉｎｅｉｎｒｏｏｔｂａｒｋｏｆＤｉｃｔａｍｎｕｓｄａｓｙｃａｒｐｕｓＴｕｒｃｚ．ＺｈｕＤａｎＮｉ,Ｘｕ...     

含翻译增强子的表达载体pSC34的构建及初步应用

１９８８年Ｏｌｉｎｓ［１］发现Ｔ７噬菌体基因１０的先导序列（Ｔ７ｇ１０Ｌ）具有明显的促进基因翻译的作用．本文构建了含Ｔ７ｇ１０Ｌ的表达载体ｐＳＣ３４,并尝试表达了几个不同类型的基因．１材料与方法１．１菌株大肠杆菌菌株ＴＡＰ１０６为本室储存．ＴＡＰ１０...