兔感觉神经特异蛋白的纯化及稳定性观察

以兔脊神经节及背根纤维为材料,通过制备匀浆,DEAE-Sephacel阴离子交换层析,高压液相凝胶过滤层析分离纯化了感觉神经特异蛋白29 ku,并进行了该蛋白的稳定性观察.     

博莱霉素-Ce(Ⅲ)对DNA的切断机理初探

根据外切核酸酶Ⅲ酶解博莱霉素-Ce(Ⅲ)［BLMA5-Ce(Ⅲ)］作用过的双链直线型DNA时, 酶解速率明显增大, 酶解产物除5′-dAMP、5′-dGMP、5′-dCMP和5′-dTMP 4种单核苷酸外, 还有其他成分存在的实验事实, 推测出BLMA5-Ce(Ⅲ)在DNA双链的特定部位沿5′→3′的方向切断磷酸二酯键, 使DNA的双链上形成多个暴露的3′-OH末端.     

高压静电场对绵羊精子存活率的影响

采用不同剂量的高压静电场处理绵羊精液,经分析发现,高压静电场对绵羊精具有激活作用。能提高绵羊精液品质,表现在适当剂量的高压静电场能显著地提高绵羊精子存活率,其中以６００ｋＶ／ｍ剂量处理效果最佳。１００ｋＶ／ｍ和３００ｋＶ／ｍ剂量对精子刺激不足,而９００ｋＶ／ｍ剂量则对精子刺激过程,导致部分精子损伤和死亡,同样达不到预期的效果。     

Formation of amino acid from urea and ammonia by sequential enzyme reaction using a microencapsulated multi-enzyme system

A microencapsulated multi-enzyme system has been used for the conversion of urea and ammonia into an amino acid, glutamate. The microencapsulated multi-enzyme system contains urease (E.C.3.5.1.5), glutamate dehydrogenase (E.C.1.4.1.3), and glucose-6-phosphate dehydrogenase (E.C.1.1.1.49). The conversion of urea into glutamate is achieved by the sequential reaction of urease and glutamate dehydrogenase; while glutamate dehydrogenase and glucose-6-phosphate dehydrogenase allow for the cyclic regeneration of NADP⁺:NADPH required for the reaction. The rate of production of glutamate is 1.3 μmole per min per ml of microcapsules. The encapsulated multi-enzyme system thus allows for the sequential enzyme reaction for the conversion of urea and ammonia into an amino acid.     

The cytolytic isolation of the cortex of the sea urchin egg

When the vitelline layer of sea urchin eggs (Lytechinus pictus) is disrupted by trypsin or dithiothreitol and the eggs are placed in an isosmotic medium devoid of Ca²⁺, cytolysis of the eggs occurs. During lysis the entire egg cortex peels off in one piece. Lysis is temperature and pH dependent and is inhibited by cytochalasin B. Cortices from unfertilized eggs contain seven major macromolecular components. A 42K-dalton component is believed to be actin, representing between 12 and 27% of the total protein. Cortices from fertilized eggs may contain between 50 and 65% actin. The actin appears to increase the strength of its attachment to the cortex after fertilization. This method of isolating the entire cortex may be useful for studying structural and enzymatic changes which may occur in the cortex during the cell cycle.     

The accessory function of murine intercellular adhesion molecule-1 in T lymphocyte activation. Contributions of adhesion and co-activation.

These studies demonstrate that the murine intercellular adhesion molecule-1 (ICAM-1) performs at least two roles in enhancing T cell activation. These two roles are evident in both of our experimental systems: with ICAM-1 expressed on the surface of transfected fibroblast cells, and with purified ICAM-1 immobilized on plastic. First, as has been documented by many investigators, ICAM-1 mediates adhesion between ICAM-1- and lymphocyte function-associated Ag-1 (LFA-1)-bearing cells. This adhesive interaction occurs even in the absence of T cell stimulation, although it is increased by addition of phorbol ester and calcium ionophore. Although ICAM-1 expression does markedly increase intercellular adhesion, the increase is significantly less than the improvement ICAM-1 expression makes in the Ag-presenting ability of MHC class II-transfected fibroblast cells. We have investigated whether this difference is due to LFA-1-mediated signaling, and we present data that demonstrates that although ICAM-1 does not deliver costimulatory signals required for T cell activation, the interaction of LFA-1 with ICAM-1 does synergize with TCR-transduced signals. This synergy is observed for ICAM-1 on live and on chemically fixed accessory cells, and for purified ICAM-1 molecules, but in all cases occurs only when the ICAM-1 and the TCR ligands are on the same surface. Finally, when the ICAM-1 is present on the surface of accessory cells, it enhances T cell activation by changing the Ag dose-dependence of the T cell, but when ICAM-1 and CD3 mAb are co-immobilized, ICAM-1 increases the peak response of the T cell without affecting the dose dependence of the response.     

Altered processing of integrin receptors during keratinocyte activation

We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells.     

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.     

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel,VDAC

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found inNeurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 µg/ml) increases the voltage dependence of VDAC channels over 2–3-fold. At higher protein concentrations (50–100 µg/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifierin vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.     

~(125)Ⅰ标记单抗LC-I与肺腺癌细胞体内外结合特性的研究

本文用~(125)Ⅰ标记LC-1进行了一些体内外实验。实验结果表明:LC-1单抗的结合常数为4.8×10~8M~(-1),LC-1针对的SPC-A_1细胞表面抗原的位点数为7.2×10~4/细胞;LC-1与LAC-122两单抗针对的抗原决定簇没有交叉;用蛋白酶和过碘酸钠处理SPC-A_1细胞,前者对LC-1的结合抑制39%,后者抑制66%;LC- 1不但有较强的体外结合靶细胞的能力,从LC-1在荷瘤裸鼠中的组织器官分布来看,LC-1与肿瘤有较高的体内亲和性,并且是特异性的结合。