Optimization of the cellular import of functionally active SH2-domain-interacting phosphopeptides

Phosphopeptides interacting with src homology 2 (SH2) domains can activate essential signaling enzymes in vitro. When delivered to cells, they may disrupt protein-protein interactions, thereby influencing intracellular signaling. We showed earlier that phosphopeptides corresponding to the inhibitory motif of Fcγ receptor IIb and a motif of the Grb2-associated binder 1 adaptor protein activate SH2-containing tyrosine phosphatase 2 in vitro. To study the ex vivo effects of these peptides, we have now compared different methods for peptide delivery: (i) permeabilization of the target cells and (ii) the use of cell-permeable vectors, which are potentially able to transport biologically active compounds into B cells. We found octanoyl-Arg₈ to be an optimal carrier for the delivery of phosphopeptides to the cells. With this strategy, the function of cell-permeable SHP-2-binding phosphopeptides was analyzed. These peptides modulated the protein phosphorylation in B cells in a dose- and time-dependent manner. Received 27 July 2006; received after revision 4 September 2006; accepted 18 September 2006     

两种织纹螺对TTX和PSP摄食转移的初步研究

织纹螺（Nassariusspp.）是沿海地区较常见的螺种之一,对其带毒途径现还存有分歧.本文选择厦门海域常见两种织纹螺,粗肋织纹螺（Nassarius nodiferus）和红带织纹螺（Nassarius succinctus）作为研究对象,以实验室生态环境下培养的塔玛亚历山大藻和月腹刺鲀内脏投喂织纹螺.采用美国分析化学家协会推荐的小白鼠的生物检测法,对织纹螺进行毒素检测.小白鼠生物检测结果表明,红带织纹螺本身不带毒性,而粗肋织纹螺本身带有毒性,麻痹性贝毒和河豚毒素两种毒素都可以通过摄食转移累积毒性.     

Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.     

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.     

Triggering of cyclin degradation in interphase extracts of amphibian eggs by cdc2 kinase

The cell cycles of early Xenopus embryos consist of a rapid succession of alternating S and M phases. These cycles are controlled by the activity of a protein kinase complex (cdc2 kinase) which contains two subunits. One subunit is encoded by the frog homologue of the fission yeast cdc2+ gene, p34cdc2 and the other is a cyclin. The concentration of cyclins follows a sawtooth oscillation because they accumulate in interphase and are destroyed abruptly during mitosis. The association of cyclin and p34cdc2 is not sufficient for activation of cdc2 kinase, however; dephosphorylation of key tyrosine and threonine residues of p34cdc2 is necessary to turn on its kinase activity. The activity of cdc2 kinase is thus regulated by a combination of translational and post-translational mechanisms. The loss of cdc2 kinase activity at the end of mitosis depends on the destruction of the cyclin subunits. It has been suggested that this destruction is induced by cdc2 kinase itself, thereby providing a negative feedback loop to terminate mitosis. Here we report direct experimental evidence for this idea by showing that cyclin proteolysis can be triggered by adding cdc2 kinase to a cell-free extract of interphase Xenopus eggs.     

猪血浆α_1—抗胰蛋白酶的紫外光谱和园二色谱

本文报告了对猪血浆α_1抗胰蛋白酶的紫外吸收光谱和园二色谱的测定结果。从紫外光谱分析得知猪α_1抗胰蛋白酶表现出典型的蛋白质吸收特征,其摩尔消光系数E_(280nm)~1％1cm 等于5.25;从(?)二色谱分析得知该蛋白质分子中合有约8％的α螺旋,50％的β-折叠,其余均为无规则卷曲构象。本测定结果对猪α_1-抗胰蛋白酶的性质、结构和功能的研究打下了基础。     

猴头菌降解木质纤维素的研究

从四株猴头菌在培养期间对木质素、半纤维素,纤维素的降解和有关酶活变化的规律进行研究。结果表明: 木质素降解量大于半纤维素降解量,半纤维素降解量大于纤维素降解量。不同菌株木质素分解活性不同,木质素分解活性强的菌株,半纤维素和纤维素的降解也较多,羧甲基纤维素酶活性和滤纸酶活性大体一致。胞外多酚氧化酶活性以培养最初期为高,后期活性很低,但子实体中的多酚氧化酶活性很高,以原基期为最高。这表明子实体中的多酚氧酶是在子实体中产生的,而不是在菌丝体中产生而后输送到子实体中去的。