Resolution of aflatoxins B1, B2, G1, and G2 by high-pressure liquid chromatography.

Aflatoxins were completely resolved as sharp peaks in the order BU-B2-G1-G2 by high-pressure liquid chromatography on a small particle (10 mum) porous silica gel column in 7-13 min (B1 through G2) by a water-saturated chloroform-cyclohexane-acetonitrile elution solvent (25+7.5+1.0), with detection by ultraviolet absorbance at 360 nm. The relationship between peak height and amount injected was linear over a 5-400 ng range for each aflatoxin. Both retention times and peak heights were highly reproducible, multiple injections of mixed standards giving coefficients of variation of 1.0-1.4% (retention time) and 1.6-2.8% (peak height) for the 4 aflatoxins. Detection was highly sensitive, with mean peak height, mm/ng, of 7.1 (B1), 6.4 (B2), 4.5 (G1), and 4.1(G2), allowing detection of 1-2 ng of each aflatoxin.     

Effect of heat treatment on the circular dichroism spectra of bovine beta-lactoglobulin A, B, and C.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated in phosphate buffer at temperatures between 40 and 94 degrees C for 10 min, cooled, and analyzed using near-UV and far-UV circular dichroism (CD). The decrease in near-UV CD intensity at 293 nm (Deltaepsilon(293)) could be analyzed in terms of a two-state model, and the stability was beta-Lg C > beta-Lg A > beta-Lg B on the basis of the midpoint temperatures for samples heated at pH 6.7 and 7.4. However, the slopes of the curves at the midpoint temperature for variant A were generally less than those for beta-Lg B and beta-Lg C, indicating that the substitution of Val (beta-Lg A) for Ala (beta-Lg B or beta-Lg C) at position 118 had altered the entropic contribution to unfolding of the protein. The changes in CD at 270 nm (Deltaepsilon(270)), an index of significant alteration to disulfide bond dihedral angles, occurred at higher temperatures than those for the Deltaepsilon(293) results. The far-UV CD showed some small changes as a consequence of heat treatment, and the shifts at 205 nm ([theta](205)) fitted a two-state model. Plotting the changes in both Deltaepsilon(293) and [theta](205) against the loss of nativelike and sodium dodecyl sulfate-monomeric protein (assessed by polyacrylamide gel electrophoresis) showed a strong 1:1 relationship between Deltaepsilon(293) or [theta](205) and the loss of nativelike beta-Lg. These results indicated that the initial irreversible stage in the heat-induced aggregation of beta-Lg (nativelike monomer to unfolded monomer) altered the chirality of the environment of Trp(19) and modified the secondary structure of beta-Lg slightly. The differences in the behavior of variants A-C were explicable on the basis of generalized electrostatic and hydrophobicity effects as well as specific amino acid effects.     

松材线虫与拟松材线虫rDNA中ITS区的比较研究

本研究利用PCR－RFLP和DNA测序技术分析了松材线虫和拟松材线虫rDNA中的ITS区。ITS区界于rDNA的18S和28S基因之间,并被5.8S基因分为ITS1和ITS2两个区。5种限制性内切酶酶切结果表明,松材线虫和拟松材线虫rDNA的ITS区存在着显著的差异;在松材线虫不同来源株系中,来自中国和日本的株系的酶切图谱一致,与来自加拿大的株系有着微小差别。对南京和日本的松材线虫及富阳的拟松材线虫株系ITS1区的序列分析显示在ITS1的280bp中,南京和日本的松材线虫株系之间,同源性高达99.7%,而来自富阳的拟松材线虫与南京和日本的松材线虫ITS1区序列的同源性相对较低,分别为90％和89.6%。     

Effect of heat treatment on bovine beta-lactoglobulin A, B, and C explored using thiol availability and fluorescence.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (T(mid)) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that beta-Lg A may denature by a different mechanism from that of beta-Lg B or beta-Lg C. The T(mid) parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the T(mid) values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) substitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.     

Chemical Principles of Environmental Pollution,B.J. Alloway and D.C. Ayres

Water, Air, &; Soil Pollution -