Sensitivity of Prestaining RNA with Ethidium Bromide Before Electrophoresis and Performance of Subsequent Northern Blots Using Heterologous DNA Probes

Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde–agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 μg/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used.     

Inhibition of x‐box binding protein 1 reduces tunicamycin‐induced apoptosis in aged murine macrophages

Endoplasmic reticulum (ER) stress is induced by the accumulation of unfolded and misfolded proteins in the ER. Although apoptosis induced by ER stress has been implicated in several aging‐associated diseases, such as atherosclerosis, it is unclear how aging modifies ER stress response in macrophages. To decipher this relationship, we assessed apoptosis in macrophages isolated from young (1.5–2 months) and aged (16–18 months) mice and exposed the cells to the ER stress inducer tunicamycin. We found that aged macrophages exhibited more apoptosis than young macrophages, which was accompanied by reduced activation of phosphorylated inositol‐requiring enzyme‐1 (p‐IRE1α), one of the three key ER stress signal transducers. Reduced gene expression of x‐box binding protein 1 (XBP1), a downstream effector of IRE1α, enhanced p‐IRE1α levels and reduced apoptosis in aged, but not young macrophages treated with tunicamycin. These findings delineate a novel, age‐dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggest an important protective role of IRE1α in aging‐associated ER stress‐induced apoptosis. This novel pathway may not only be important in our understanding of longevity, but may also have important implications for pathogenesis and potential treatment of aging‐associated diseases in general.     

Emission and Control Characteristics for Incineration of Sedum Plumbizincicola Biomass in a Laboratory-Scale Entrained Flow Tube Furnace

Experiments were conducted to investigate and control pollutant emission from incineration of Sedum plumbizincicola plants on a laboratory scale using an entrained flow tube furnace. Without control technologies, the flue gas contained 0.101 mg Nm^?3 of Cd, 46.4 mg Nm^?3 of Zn, 553 mg Nm^?3 of NOx, 131 pg Nm^?3 of polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran (PCDD/Fs) and 35.4 mg Nm^?3 of polycyclic aromatic hydrocarbons (PAHs). In pollutants control experiments. Al₂O₃, CaO, and kaolin were compared as adsorbents and activated carbon was used as an end-of-pipe method for the capture of pollutants. Kaolin, the most effective of the three adsorbents, removed 91.2% of the Cd in flue gas. While 97.6% of the Cd and 99.6% of the PAHs were removed by activated carbon. Incineration may therefore be regarded as a viable option for the safe disposal of the biomass of the zinc and cadmium hyperaccumulator species S. plumbizincicola.     

Large enhancement of oscillating chemiluminescence with [Ru(bpy)3]2+‐catalyzed Belousov–Zhabotinsky reaction in the presence of tri‐n‐propylamine

Oscillating chemiluminescence enhanced by the addition of tri‐n‐propylamine (TPrA) to the typical Belousov–Zhabotinsky (BZ) reaction system catalyzed by ruthenium(II)tris(2.2'‐bipyridine)(Ru(bpy)₃²⁺) was investigated using a luminometry method. The [Ru(bpy)₃]²⁺/TPrA system was first used as the catalyst for a BZ oscillator in a closed system, which exhibited a shorter induction period, higher amplitude and much more stable chemiluminescence (CL) oscillation. The effects of various concentrations of TPrA, oxygen and nitrogen flow rate on the oscillating behavior of this system were examined. In addition, the CL intensity of the [Ru(bpy)₃]²⁺/TPrA–BZ system was found to be inhibited by phenol, thus providing a way for use of the BZ system in the determination of phenolic compounds. Moreover, the possible mechanism of the oscillating CL reaction catalyzed by [Ru(bpy)₃]²⁺/TPrA and the inhibition effects of oxygen and phenol on this oscillating CL system were considered. Copyright © 2012 John Wiley & Sons, Ltd.     

Minocycline attenuates mechanical allodynia and expression of spinal NMDA receptor 1 subunit in rat neuropathic pain model

Recent studies have indicated that minocycline, a microglia inhibitor, could potentially be used as an antinociceptive agent in pain management, although the underlying mechanisms remain elusive. In this study, we investigated the extent to which minocycline could influence pain behavior in association with the expression of the N-methyl-d-aspartic acid receptor 1 (NMDAR1) in a rat L5 spinal nerve ligation (SNL) model. We observed that the intrathecal injection of minocycline significantly attenuated mechanical allodynia in a rat SNL model from day 1 postinjection and persisted for at least 18 days. We also observed that the expression of NMDAR1 was increased in the spinal dorsal horn at 8 days after SNL, which could be partly inhibited through the intrathecal injection of minocycline. These findings suggest that the attenuation of allodynia in the SNL model following minocycline administration might be associated with the inhibited expression of NMDAR1 and, therefore, might play an important role in the minocycline-mediated antinociception.     

Development and Application of Microsatellites in Candidate Genes Related to Wood Properties in the Chinese White Poplar (Populus tomentosa Carr.)

Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are often preferred over random genomic markers because they represent variation in gene coding and/or regulatory regions. We characterized 544 genic SSR loci derived from 138 candidate genes involved in wood formation, distributed throughout the genome of Populus tomentosa, a key ecological and cultivated wood production species. Of these SSRs, three-quarters were located in the promoter or intron regions, and dinucleotide (59.7%) and trinucleotide repeat motifs (26.5%) predominated. By screening 15 wild P. tomentosa ecotypes, we identified 188 polymorphic genic SSRs with 861 alleles, 2–7 alleles for each marker. Transferability analysis of 30 random genic SSRs, testing whether these SSRs work in 26 genotypes of five genus Populus sections (outgroup, Salix matsudana), showed that 72% of the SSRs could be amplified in Turanga and 100% could be amplified in Leuce. Based on genotyping of these 26 genotypes, a neighbour-joining analysis showed the expected six phylogenetic groupings. In silico analysis of SSR variation in 220 sequences that are homologous between P. tomentosa and Populus trichocarpa suggested that genic SSR variations between relatives were predominantly affected by repeat motif variations or flanking sequence mutations. Inheritance tests and single-marker associations demonstrated the power of genic SSRs in family-based linkage mapping and candidate gene-based association studies, as well as marker-assisted selection and comparative genomic studies of P. tomentosa and related species.     

Genetic and physical fine mapping of a multilocular gene Bjln1 in Brassica juncea to a 208-kb region

Most of the germplasm resources in Brassica juncea produce silique with only two locules, whereas a few varieties can produce silique with three or four locules. The increase in locule number in B. juncea has been shown to cause an increase in the number of seeds per silique, resulting in an increase in the yield per plant. Thus, the development of high-locule-number varieties may be an effective way of improving the yield of B. juncea. Duoshi, a B. juncea landrace originating from the Qinghai–Tibetan plateau, produces silique with 3–4 locules. Genetic analysis has shown that the high-locule-number trait in Duoshi is determined by two recessive genes, tentatively designated as Bjln1 and Bjln2. For fine mapping of the Bjln1 gene, a BC3 population was developed from the cross between Duoshi (multilocular parent) and Xinjie (bilocular parent). Using a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis, only two AFLP markers linked to Bjln1 were identified. Preliminary linkage analysis showed that the two AFLP markers were located on the same side of Bjln1. Blast analysis revealed that the sequences of the two AFLP markers had homologues on Scaffold000019 at the bottom of B. rapa A7. Using the results of linkage analysis and BlastN searches, simple sequence repeat (SSR) markers were subsequently developed based on the sequence information from B. rapa A7. Seven SSR markers were eventually identified, of which ln 8 was co-segregated with Bjln1. ln 7 and ln 9, the closest flanking markers, were mapped at 2.0 and 0.4 cM distant from the Bjln1 gene, respectively. The SSR markers were cloned, sequenced and mapped on A7 of B. rapa (corresponding to J7 in the A genome of B. juncea). The two closest flanking markers, ln 7 and ln 9, were mapped within a 208-kb genomic region on B. rapa A7, in which the Bjln1 gene might be included. The present study may facilitate cloning of the Bjln1 gene as well as the selection process for developing multilocular varieties in B. juncea by marker-assisted selection and genetic engineering.     

The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.