应用环磷酰胺研制细胞表面抗原的单克隆抗体

利用环磷酰胺和杂交瘤技术 ,成功制备了 1株稳定分泌抗人CD80 分子单克隆抗体的杂交瘤细胞。实验结果表明 ,该单抗腹水效价为 1 0 6 ,属IgG1亚类 ,特异性强 ,为今后的应用打下了基础。     

猪瘟病毒p80基因NTPase/RNA解旋酶功能区在E.coli中的表达

采用PCR技术扩增出猪瘟病毒GXYL株和C株p80基因的NTPase／RNA解旋酶功能区(sGXNS3和sCNS3),将其克隆到表达载体pET-30a( )中,获得重组质粒pET—sGXNS3和pET～sCNS3。PCR、酶切和序列分析鉴定目的基因插入位置、方向和读码框完全正确。1．0mmol／L IPTG诱导得到分子量为26kD的目的蛋白。Western blot检测表明,表达的目的蛋白能被CSFV阳性血清识别。     

KTN80 confers precision to microtubule severing by specific targeting of katanin complexes in plant cells

The microtubule (MT)‐severing enzyme katanin triggers dynamic reorientation of cortical MT arrays that play crucial functions during plant cell morphogenesis, such as cell elongation, cell wall biosynthesis, and hormonal signaling. MT severing specifically occurs at crossover or branching nucleation sites in living Arabidopsis cells. This differs from the random severing observed along the entire length of single MTs in vitro and strongly suggests that a precise control mechanism must exist in vivo. However, how katanin senses and cleaves at MT crossover and branching nucleation sites in vivo has remained unknown. Here, we show that the katanin p80 subunit KTN80 confers precision to MT severing by specific targeting of the katanin p60 subunit KTN1 to MT cleavage sites and that KTN1 is required for oligomerization of functional KTN80–KTN1 complexes that catalyze severing. Moreover, our findings suggest that the katanin complex in Arabidopsis is composed of a hexamer of KTN1–KTN80 heterodimers that sense MT geometry to confer precise MT severing. Our findings shed light on the precise control mechanism of MT severing in plant cells, which may be relevant for other eukaryotes.     

Structural Basis of High‐Affinity Nuclear Localization Signal Interactions with Importin‐α

Classical nuclear localization signals (cNLSs), comprising one (monopartite cNLSs) or two clusters of basic residues connected by a 10–12 residue linker (bipartite cNLSs), are recognized by the nuclear import factor importin‐α. The cNLSs bind along a concave groove on importin‐α; however, specificity determinants of cNLSs remain poorly understood. We present a structural and interaction analysis study of importin‐α binding to both designed and naturally occurring high‐affinity cNLS‐like sequences; the peptide inhibitors Bimax1 and Bimax2, and cNLS peptides of cap‐binding protein 80. Our data suggest that cNLSs and cNLS‐like sequences can achieve high affinity through maximizing interactions at the importin‐α minor site, and by taking advantage of multiple linker region interactions. Our study defines an extended set of binding cavities on the importin‐α surface, and also expands on recent observations that longer linker sequences are allowed, and that long‐range electrostatic complementarity can contribute to cNLS‐binding affinity. Altogether, our study explains the molecular and structural basis of the results of a number of recent studies, including systematic mutagenesis and peptide library approaches, and provides an improved level of understanding on the specificity determinants of a cNLS. Our results have implications for identifying cNLSs in novel proteins.     

Possible Mechanism of Dantrolene Stabilization of Cultured Neuroblastoma Cell Plasma Membranes

Abstract: Some reports have suggested that dantrolene interacts directly with the membrane bilayer. We investigated effects of dantrolene on changes in membrane properties induced by compound 48/80 (C48/80), a membrane stimulator. The addition of C48/80 for 1 min elicited a rapid, dose-dependent Ca²⁺ influx, which was reduced to 14% by the absence of external Ca²⁺. Dantrolene inhibited the C48/80-induced increase in Ca²⁺ permeability of plasma membranes in a concentration-dependent manner (0.33–10 µ M , IC₅₀ value was 5 µ M ). We next examined C48/80-induced changes in structural and dynamic membrane properties by electron spin resonance (ESR). The ratio h ₀/ h ₋₁ was determined to evaluate membrane fluidity. C48/80 increased the membrane fluidity in a concentration-dependent manner (0.1–0.56 mg/ml). Dantrolene (10 µ M ) itself did not change the membrane fluidity, but it significantly reduced the C48/80-induced increase in membrane fluidity (0.56 mg/ml). Moreover, the C48/80-induced increase in fluidity was dependent on extracellular Ca²⁺. We conclude that dantrolene protects neuroblastoma cell plasma membrane from C48/80-induced membrane perturbation, which causes Ca²⁺ influx and an increase in membrane fluidity. These findings strongly suggest that dantrolene directly stabilizes the neuronal plasma membrane.     

Effects of hormones (calcitonin, GIP) and pharmacological antagonists (ranitidine and famotidine) on isolated rat parietal cells

The rationale for the present study was to compare calcitonin and gastric inhibitory polypeptide (GIP) versus two histamine H2 receptor antagonists with respect to their potency of inhibiting parietal cell functions. Adenylate cyclase activity and acid production ([14C]aminopyrine uptake) of isolated rat parietal cells were stimulated by histamine. At 10(-7) and 10(-6) mol/l, calcitonin and GIP reduced the response to histamine by 10-20% following noncompetitive kinetics. Ranitidine and famotidine (MK 208) inhibited the response to histamine by about 50% at 10(-7)-10(-6) mol/l, and at 10(-5) mol/l abolished the histamine effect. On a molar basis famotidine turned out to be 6 times more potent than ranitidine. Both antagonists revealed competitive kinetics. Our data suggest direct inhibition of the parietal cells by the tested compounds which were shown to interfere at the adenylate cyclase cAMP system or at the histamine H2 receptor. However, compared to the histamine H2 receptor antagonists, hormonal inhibition is less pronounced and mediated by a different mechanism.     

Mechanisms of enhanced antiglioma efficacy of polysorbate 80‐modified paclitaxel‐loaded PLGA nanoparticles by focused ultrasound

The presence of blood‐brain barrier (BBB) greatly limits the availability of drugs and their efficacy against glioma. Focused ultrasound (FUS) can induce transient and local BBB opening for enhanced drug delivery. Here, we developed polysorbate 80‐modified paclitaxel‐loaded PLGA nanoparticles (PS‐80‐PTX‐NPs, PPNP) and examined the enhanced local delivery into the brain for glioma treatment by combining with FUS. Our result showed PPNP had good stability, fast drug release rate and significant toxicity to glioma cells. Combined with FUS, PPNP showed a stronger BBB permeation efficiency both in the in vitro and in vivo BBB models. Mechanism studies revealed the disrupted tight junction, reduced P‐glycoprotein expression and ApoE‐dependent PS‐80 permeation collectively contribute to the enhanced drug delivery, resulting in significantly stronger antitumour efficacy and longer survival time in the tumour‐bearing mice. Our study provided a new strategy to efficiently and locally deliver drugs into the brain to treat glioma.     

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding

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Effective production of biologically active water-soluble β-1,3-glucan by a coupled system of Agrobacterium sp. and Trichoderma harzianum

Water-soluble β-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10?g?L^?1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis. A two-stage glucose and DO control strategy was optimal for w-glucan production. At the T. harzianum cell growth stage, the optimal glucose flow rate and agitation speed were 2.0?g?L^?1 hr^?1 and 600?rpm, respectively, and at the w-glucan production stage, they were 0.5?g?L^?1 hr^?1 and 400?rpm, respectively. W-glucan production reached 17.31?g?L^?1, with a degree of polymerization of 19–25. Furthermore, w-glucan at high concentrations exhibited anti-tumor activity against MCF-7, HepG2, and Hela cancer cells in vitro. This study provides a novel, cost-effective, eco-friendly, and efficient microbial fermentation method for the direct production of biologically active w-glucan.