蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

CD4+ T cell response to the human acetylcholine receptor alpha subunit in myasthenia gravis. A study with synthetic peptides

The production of antinicotinic acetylcholine receptor (AcChR) antibodies in myasthenia gravis (MG) is modulated by specific Th (CD4+) lymphocytes that can recognize epitopes on the denatured AcChR alpha subunit. Thirty-two overlapping synthetic peptides corresponding to the complete sequence of human AcChR alpha subunit were used to investigate the anti-alpha subunit response of unselected lymphocytes and of CD8(+)-depleted, CD4(+)-enriched lymphocytes from the blood of nine MG patients and from four healthy controls. One subject was a newly diagnosed MG patient that was tested three times after the development of the disease. An anti-AcChR response of the CD4(+)-enriched cells was present that could be detected only after removal of the CD8+ population and that seems to be related to the clinical conditions of the patient. The high basal rate of the cell proliferation of the unselected unstimulated blood lymphocytes and the normal basal rate observed for the CD8(+)-depleted population suggested the presence of activated CD8+ cells. The study of surface markers of the T cells confirmed the existence of activated CD8+ and CD4+ cells in numbers correlated with the severity of the disease and the results of the in vitro response of the T cells. The anti-AcChR activity of the CD4+ cells in MG may be a useful marker of the activity of the disease and it seems to be influenced by activated CD8+ cells present in the patients' blood.     

Taxol binds to cellular microtubules

Taxol is a low molecular weight plant derivative which enhances microtubule assembly in vitro and has the unique ability to promote the formation of discrete microtubule bundles in cells. Tritium-labeled taxol binds directly to microtubules in vitro with a stoichiometry approaching one (Parness, J., and S. B. Horwitz, 1981, J. Cell Biol. 91:479-487). We now report studies in cells on the binding of [3H]taxol and the formation of microtubule bundles. [3H]Taxol binds to the macrophagelike cell line, J774.2, in a specific and saturable manner. Scatchard analysis of the specific binding data demonstrates a single set of high affinity binding sites. Maximal binding occurs at drug concentrations which produce maximal growth inhibition. Conditions which depolymerize microtubules in intact and extracted cells as determined by tubulin immunofluorescence inhibit the binding of [3H]taxol. This strongly suggests that taxol binds specifically to cellular microtubules. Extraction with 0.1% Nonidet P-40 or depletion of cellular ATP by treatment with 10 mM NaN3 prevents the characteristic taxol-induced bundle formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there must be specific cellular mechanisms which are required for bundle formation, in addition to the direct binding of taxol to cytoplasmic microtubules.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient

A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix.