二氧化硫代谢衍生物对大鼠海马CA1区神经元钠电流的影响

实验采用全细胞膜片钳技术 ,研究了SO2 代谢衍生物亚硫酸钠和亚硫酸氢钠 (两者分子比为 3∶1)对大鼠海马CA1区神经元钠电流的影响。结果表明 ,SO2 代谢衍生物可剂量依赖性地增大钠电流 ,剂量为 10和 10 0μmol/L时 ,钠电流分别增大 5 0 .5 9± 19.0 8%和 82 .0 6± 18.5 1%(n =15 ) ;此外还与电压呈依赖性关系 ,但不具有频率依赖性 ;10 μmol/LSO2 代谢衍生物不影响钠电流的激活过程 ,却非常显著地影响其失活过程 ,作用前后的半数失活电压分别为 - 6 9.71± 4.6 7和 - 5 3.2 7± 4.95mV (n =10 ,P <0 .0 1) ,但不改变失活曲线的斜率因子。实验结果提示 ,SO2 衍生物具有类似神经毒物的作用 ,大气SO2 污染可能与一些中枢神经系统疾病的发生有关。     

Efficient regeneration of <Emphasis Type="Italic">Sphagnum fallax</Emphasis> from isolated protoplasts

Summary While the in vitro clonal propagation of peat mosses (Sphagnaceae) in bioreactors has been established since the late 1980s, it has never been possible to regenerate Sphagnum species from isolated protoplasts, which is a key step towards the production of closely defined genetically modified clones. The present study describes an efficient protocol for protoplast isolation and regeneration of Sphagnum fallax. Protoplast survival rates of over 50% and regeneration rates of up to 20% were achieved by using excised capitulum buds as starting material and by co-cultivating Sphagnum protoplasts with protoplasts from a chlorophyll-deficient Solanum hybrid clone. Besides the effects of nutrient components and differential osmotic readjustment of the regenerant cell clusters, the interference of unique Sphagnum phenolics, sphagnum acid and hydroxybutenolide, with protoplast isolation efficiency is demonstrated.     

A water-stable protected isocyanate glass array substrate

We describe the performance of a new glass attachment chemistry for arrays that is particularly well suited to attachment of small molecules, such as peptides. The attachment chemistry is a protected isocyanate (PI) group. Isocyanate groups are well suited to serving as a glass coating for arrays, in that they are highly reactive with many different types of biological compounds. However, they are generally so reactive as to be unstable. The new feature of the PI slide coating is its stability. It can withstand immersion in water without loss of reactivity and has at least a 1-year shelf life. The high reactivity of the PI group results in a rapid coupling reaction (< 15min) and is particularly useful for attaching small molecules, such as peptides. Since isocyanates bind to both amines (forming a urea linkage) and hydroxyl groups (forming a carbamate bond), we tested the ability of the PI coating to bind to a wide variety of compounds. We found that the PI slide coating can directly attach to peptides, proteins, carbohydrates, lipooligosaccharides, and DNA. The sensitivity of detection for these compounds is comparable to that of other previously published array substrates.     

Versatile protein microarray based on carbohydrate-binding modules

Non-DNA microarrays, such as protein, peptide and small molecule microarrays, can potentially revolutionize the high-throughput screening tools currently used in basic and pharmaceutical research. However, fundamental obstacles remain that limit their rapid and widespread implementation as an alternative bioanalytical approach. These include the prerequisite for numerous proteins in active and purified form, ineffectual immobilization strategies and inadequate means for quality control of the considerable numbers of multiple reagents. This study describes a simple yet efficient strategy for the production of non-DNA microarrays, based on the tenacious affinity of a carbohydrate-binding module (CBM) for its three-dimensional substrate, i.e., cellulose. Various microarray formats are described, e.g., conventional and single-chain antibody microarrays and peptide microarrays for serodiagnosis of human immunodeficiency virus patients. CBM-based microarray technology overcomes many of the previous obstacles that have hindered fabrication of non-DNA microarrays and provides a technically simple but effective alternative to conventional microarray technology.     

Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination

Protein analysis techniques are developing fast due to the growing number of proteins obtained by recombinant DNA techniques. In the present paper we compare selected techniques, which are used for protein sizing, quantitation and molecular weight determination: sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip or microfluidics technology (LoaC), size exclusion chromatography (SEC) and mass spectrometry (MS). We compare advantages and limitations of each technique in respect to different application areas, analysis time, protein sizing and quantitation performance.     

三羟异黄酮对豚鼠心室肌细胞L-型钙通道电流的影响

本实验用全细胞膜片钳技术观察三羟异黄酮(genistein,GST)对豚鼠心室肌细胞L-钙通道电流(ICa、L)的影响。结果如下:(1)GST(10、50、100 μmol/L)可浓度依赖性地降低ICa,L(n=6,P<0.01)。GST的非活性结构类似物daidzein(100μmol/L),在同一浓度范围对ICa,L没有影响(n=5,P>0.05)。(2)GST使I-V曲线上移,但对ICa,L的电压依赖特征和最大激活电压无明显影响。(3)GST对ICa,L的激活动力学特性也无影响,但可使钙电流稳态失活曲线左移。V0.5从对照的-28.6±0.6 mV变为-32.8±1.1mV,κ值从对照的5.8±0.5 mV升至6.5±0.9 mV(n=6,P<0.05)。(4)GST明显使复活曲线右移,从而使ICa,L从失活状态下恢复明显减慢(n=7,P<0.01)。(5)酪氨酸磷酸酶抑制剂正钒酸钠(1 mmol/L)显著对抗GST引起的ICa,L抑制效应(n=6,P<0.01)。根据以上结果得出的结论是:GST抑制ICa,L加速钙通道失活和钙通道在失活状态下恢复减慢;GST对ICa,L的这种抑制作用与蛋白酪氨酸激酶(PTK)抑制有关。     

Crystal structure determination of thymoquinone by high-resolution X-ray powder diffraction

The crystal structure of 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) and its thermal behavior--as necessary physical and chemical properties--were determined in order to enhance the current understanding of thymoquinone chemical action by using high resolution x-ray powder diffraction, Fourier transform infrared spectroscopy (FTIR), and 3 thermo-analytical techniques thermogravimetric analysis (TGA), differential thermal analysis (DTA), and differential scanning calorimetry (DSC). The findings obtained with high-resolution x-ray powder diffraction and molecular location methods based on a simulated annealing algorithm after Rietveld refinement showed that the triclinic unit cell was a = 6.73728(8) A, b = 6.91560(8) A, c = 10.4988(2) A, alpha = 88.864(2) degrees, beta = 82.449(1) degrees, gamma = 77.0299(9) degrees; cell volume = 472.52(1) A3, Z = 2, and space group P1. In addition, FTIR spectrum revealed absorption bands corresponding to the carbonyl and C-H stretching of aliphatic and vinylic groups characteristically observed in such p-benzoquinones. Also, a chemical decomposition process starting at 65 degrees C and ending at 213 degrees C was noted when TGA was used. DSC allowed for the determination of onset at 43.55 degrees C and a melting enthalpy value of DeltaH(m) = 110.6 J/g. The low value obtained for the fusion point displayed a van der Waals pattern for molecular binding, and the thermograms performed evidence that thymoquinone can only be found in crystalline triclinic form, as determined by DRX methods.     

Binding of mineral elements to locust bean gum influences availability in vitro

Experiments were conducted to evaluate the extent to which element binding of locust bean gum (LBG) affects the availability of calcium, iron, and zinc in the gut. Infant formula was supplemented with increasing amounts of LBG and subjected to an intraluminal digestion procedure. Element binding was measured by eliminating the complexes by twofold centrifugation. Availability of the elements was determined using a validated continuous-flow dialysis technique. Elemental content of the samples, supernatants, and dialysates was analyzed with validated atomic absorption spectrometry. LBG provided small amounts of intrinsic calcium (1.13 ± 0.02 mg/g) and trace amounts of iron (0.02 ± 0.00 mg/g) and zinc (0.01 ± 0.00 mg/g), which were strongly bound to the LBG molecule (respectively: 76.6 ± 3.3%, 83.4 ± 1.2%, 96.7 ± 6.6%). Correlation analysis, between percent element bound by LBG after centrifugation and percent trapped after dialysis, yielded significant correlation only for the data of zinc (r=0.93). For calcium and iron, no correlation could be demonstrated; however, for iron a similar trend was observed. These findings suggest that element binding of LBG has a major influence on the availability of zinc and maybe of iron. For calcium, other factors might also be involved, affecting availability.     

Assessment of the food safety issues related to genetically modified foods

International consensus has been reached on the principles regarding evaluation of the food safety of genetically modified plants. The concept of substantial equivalence has been developed as part of a safety evaluation framework, based on the idea that existing foods can serve as a basis for comparing the properties of genetically modified foods with the appropriate counterpart. Application of the concept is not a safety assessment per se, but helps to identify similarities and differences between the existing food and the new product, which are then subject to further toxicological investigation. Substantial equivalence is a starting point in the safety evaluation, rather than an endpoint of the assessment. Consensus on practical application of the principle should be further elaborated. Experiences with the safety testing of newly inserted proteins and of whole genetically modified foods are reviewed, and limitations of current test methodologies are discussed. The development and validation of new profiling methods such as DNA microarray technology, proteomics, and metabolomics for the identification and characterization of unintended effects, which may occur as a result of the genetic modification, is recommended. The assessment of the allergenicity of newly inserted proteins and of marker genes is discussed. An issue that will gain importance in the near future is that of post-marketing surveillance of the foods derived from genetically modified crops. It is concluded, among others that, that application of the principle of substantial equivalence has proven adequate, and that no alternative adequate safety assessment strategies are available.