皮质酮对高钾诱导PC12细胞内钙升高的非基因组调节作用

目的：分析皮质酮快速抑制高钾诱导PC12细胞内钙升高的机制。方法：使用荧光影像系统,实时检测细胞内钙变化。结果：①在预处理PC12细胞5min后,皮质酮可呈量－效关系抑制高钾诱导的PC12细胞内钙升高。②牛血清白蛋白耦联的皮质酮（B－BSA）可模拟皮质酮快速抑制高钾诱导PC12细胞内钙升高的作用,并呈现量－效关系。③糖皮质激素的细胞内受体拮抗剂RU38486对皮质酮快速抑制效应无明显拮抗作用。④蛋白合成抑制剂放线菌酮预处理细胞3h后,皮质酮对高钾诱导PC12内钙升高仍有较强抑制作用。结论：①皮质酮的作用点位于细胞膜上。②皮质酮的快速作用不直接依赖细胞内蛋白合成和不依赖细胞内糖皮质激素受体。③皮质酮对高钾诱导PC12内钙升高的快速抑制作用属非基因组作用。     

IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth

Plants are the principal source of iron in most diets, yet iron availability often limits plant growth. In response to iron deficiency, Arabidopsis roots induce the expression of the divalent cation transporter IRT1. Here, we present genetic evidence that IRT1 is essential for the uptake of iron from the soil. An Arabidopsis knockout mutant in IRT1 is chlorotic and has a severe growth defect in soil, leading to death. This defect is rescued by the exogenous application of iron. The mutant plants do not take up iron and fail to accumulate other divalent cations in low-iron conditions. IRT1-green fluorescent protein fusion, transiently expressed in culture cells, localized to the plasma membrane. We also show, through promoter::beta-glucuronidase analysis and in situ hybridization, that IRT1 is expressed in the external cell layers of the root, specifically in response to iron starvation. These results clearly demonstrate that IRT1 is the major transporter responsible for high-affinity metal uptake under iron deficiency.     

Modulation of alternative pre-mRNA splicing in vivo by pinin

Pre-mRNA splicing occurs in a large macromolecular RNA-protein complex called the spliceosome. The major components of the spliceosome include snRNP and SR proteins. We have previously identified an SR-like protein, pinin (pnn), which is localized not only in nuclear speckles but also at desmosomes. The nuclear localization of pnn is a dynamic process because pnn can be found not only with SR proteins in nuclear speckles but also in enlarged speckles following treatment of cells with RNA polymerase II inhibitors, DRB, and alpha-amanitin. Using adenovirus E1A and chimeric calcitonin/dhfr construct as a splicing reporter minigene in combination with cellular cotransfection, we found that pnn regulates alternative 5(') and 3(') splicing by decreasing the use of distal splice sites. Regulation of 5(') splice site choice was also observed for RNPS1, a general splicing activator that interacts with pnn in nuclear speckles. The regulatory ability of pnn in alternative 5(') splicing, however, was not dependent on RNPS1 and a pnn mutant, lacking the N-terminal 167 amino acids, behaved like a dominant negative species, inhibiting E1A splicing when applied in splicing assays. These results provide direct evidence that pnn functions as a splicing regulator which participates itself directly in splicing reaction or indirectly via other components of splicing machinery.     

A rapid,nongenomic action of glucocorticoids in rat B103 neuroblastoma cells

We report here a new example in which glucocorticoids (GCs) acted in a rapid, nongenomic way. In rat B103 neuroblastoma cells, 5-hydroxytryptamine (5-HT) was found to evoke an immediate rise in intracellular free calcium concentration ([Ca(2+)](i)). Pre-incubation of B103 cells for 5 min with corticosterone (B) or bovine serum albumin-conjugated corticosterone (B-BSA) concentration-dependently (10(-4)-10(-8) M) inhibited the peak increments in [Ca(2+)](i). Cortisol and dexamethasone had a similar effect, while deoxycorticosterone and cholesterol were ineffective. This rapid inhibitory effect of corticosterone could be mimicked by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and abolished completely by PKC inhibitors Ro31-8220 or GF-109203X. Neither pertussis toxin (PTX) nor nuclear GC receptor (GR) antagonist RU38486 influenced the rapid action of B. Our results suggest that GCs can modulate the 5-HT-induced Ca(2+) response in B103 cells in a membrane-initiated, nongenomic, and PKC-dependent manner.     

Antibodies in proteomics II: screening,high-throughput characterization and downstream applications

There are many ways in which the use of antibodies and antibody selection can be improved and developed for high-throughput characterization. Standard protocols, such as immunoprecipitation, western blotting and immunofluorescence, can be used with antibody fragments generated by display technologies. Together with novel approaches, such as antibody chips and intracellular immunization, these methods will yield useful proteomic data following adaptation of the protocols for increased reliability and robustness. To date, most work has focused on the use of standard, well-characterized commercial antibodies. Such protocols need to be adapted for broader use, for example, with antibody fragments or other binders generated by display technologies, because it is unlikely that traditional approaches will provide the required throughput.     

The reduction of Raf-1 protein by phosphorothioate ODNs and siRNAs targeted to the same two mRNA sequences

Two sets of 20-mer phosphorothioate-modified oligodeoxynucleotide DNAs (sODN) and 21-mer or 22-mer small interfering RNAs (siRNAs), targeted to the same coding sites in raf-1 mRNA, were compared for their abilities to reduce the amount of endogenously expressed Raf-1 protein in T24 cells. The amount of Raf-1 protein was monitored by careful quantitation of Western blots. We found that the siRNAs were somewhat less effective than the S-ODNs in reducing the Raf-1 protein level 20 hours after a 4-hour transfection. The siRNA duplexes were characterized by circular dichroism (CD) spectra, and melting temperatures (Tm) were obtained for the siRNA duplexes and DNA x RNA hybrids formed by the S-ODNs. The S-ODNs differed in their effectiveness, the S-ODN that formed the more stable hybrid being the more effective in reducing the Raf-1 protein level, but the two siRNAs were equally effective despite a difference in Tm of about 20 degrees C. Finally, the siRNAs and S-ODNs had a comparable nonspecific effect on a nontargeted (Bcl-2) protein. Our data add to others in the literature that show it can be difficult to select siRNAs that are more effective than antisense ODNs in downregulating endogenously expressed proteins.