Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides

Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.     

Cellular Uptake and Antitumor Activity of DOX-hyd-PEG-FA Nanoparticles

A PEG-based, folate mediated, active tumor targeting drug delivery system using DOX-hyd-PEG-FA nanoparticles (NPs) were prepared. DOX-hyd-PEG-FA NPs showed a significantly faster DOX release in pH 5.0 medium than in pH 7.4 medium. Compared with DOX-hyd-PEG NPs, DOX-hyd-PEG-FA NPs increased the intracellular accumulation of DOX and showed a DOX translocation from lysosomes to nucleus. The cytotoxicity of DOX-hyd-PEG-FA NPs on KB cells was much higher than that of free DOX, DOX-ami-PEG-FA NPs and DOX-hyd-PEG NPs. The cytotoxicity of DOX-hyd-PEG-FA NPs on KB cells was attenuated in the presence of exogenous folic acid. The IC₅₀ of DOX-hyd-PEG-FA NPs and DOX-hyd-PEG NPs on A549 cells showed no significant difference. After DOX-hyd-PEG-FA NPs were intravenously administered, the amount of DOX distributed in tumor tissue was significantly increased, while the amount of DOX distributed in heart was greatly decreased as compared with free DOX. Compared with free DOX, NPs yielded improved survival rate, prolonged life span, delayed tumor growth and reduced the cardiotoxicity in tumor bearing mice model. These results indicated that the acid sensitivity, passive and active tumor targeting abilities were likely to act synergistically to enhance the drug delivery efficiency of DOX-hyd-PEG-FA NPs. Therefore, DOX-hyd-PEG-FA NPs are a promising drug delivery system for targeted cancer therapy.     

Construction of a recombinant BHV-1 expressing the VP1 gene of foot and mouth disease virus and its immunogenicity in a rabbit model

Foot-and-mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle. Using bovine herpesvirus type 1 (BHV-1) as a gene delivery vector for development of live-viral vaccines has gained widespread interest. In this study, a recombinant BHV-1 was constructed by inserting the synthetic FMDV (O/China/99) VP1 gene in the the gE locus of BHV-1 genome under the control of immediately early gene promoter of human cytomegalovirus (phIE CMV) and bovine growth hormone polyadenylation (BGH polyA) signal. After homologous recombination and plaque purification, a recombinant virus named BHV-1/gE⁻/VP1 was acquired and identified. The immunogenicity was confirmed in a rabbit model by virus neutralization test and enzyme-linked immunosorbent assay (ELISA). The result indicated that the BHV-1/gE⁻/VP1 has the potential for being developed as a bivalent vaccine for FMD and IBR.     

STAT6 ASODN对哮喘小鼠脾淋巴细胞影响的实验研究

目的研究STAT6反义寡核苷酸对哮喘小鼠脾淋巴细胞的影响作用。方法实验细胞分组:正常鼠空白组(A组)、正常鼠OVA组(B组)、哮喘空白组(C组)、哮喘OVA组(D组)、哮喘治疗组(E组)。正常设计并人工合成一段互补于小鼠STAT6 mRNA翻译起始区271-290的反义寡核苷酸片段,全链硫代修饰。用卵白蛋白和氢氧化铝复制哮喘模型,用淋巴细胞分离液分离脾淋巴细胞,进行体外培养并导入由阳离子脂质体转染剂Geneshuttle携带的反义寡核苷酸,观察反义寡核苷酸的转染对脾淋巴细胞STAT6蛋白表达水平及细胞培养上清中IL-4分泌水平的影响。免疫细胞化学观察脾淋巴细胞中STAT6蛋白的表达水平,同时采用酶联免疫吸附(ELISA)法测定脾细胞培养上清液中IL-4的浓度。结果D组细胞STAT6蛋白表达明显高于其余各组,均具有显著性差异(P均<0.01),STAT6 ASODN转染后,E组细胞该蛋白的表达量明显下降(P<0.01);D组脾淋巴细胞培养上清中IL-4分泌水平明显高于其余各组,均具有显著性差异(P均<0.01);STAT6 ASODN转染后,E组培养上清中IL-4分泌水平显著低于D组(P<0.01)。结论STAT6 ASODN可特异性抑制哮喘鼠脾淋巴细胞中STAT6蛋白的表达,并可特异性抑制脾淋巴细胞中IL-4的分泌,为反义基因技术治疗哮喘提供了依据。     

Hydrophobic chromatography of the HL-60 cellular fraction co-binding with hexamethylene bisacetamide

Methods of separating N-acetyl-1,6-diaminohexane (NADAH) and its immobilization to diol-silica have been developed. Hexamethylene bisacetamide (HMBA) and its metabolite NADAH are used as inducers of leukemia cell differentiation. The inducing mechanism of HMBA is still not clear. Experiments show that HMBA and NADAH undergo relatively strong hydrophobic reactions and do not readily undergo ion-exchange with the proteins of the cytosolic fraction of HL-60 cells during immobilization of NADAH; the retention time of the proteins was longer than that of the phosphatides. These results show that the adsorption of HMBA and NADAH to proteins was higher than that to phosphatides. The expected biospecific receptor binding with HMBA has not been found.     

Nitrogen and phosphorus addition differentially enhance seed production of dominant species in a temperate steppe

Previous studies have demonstrated changes in plant growth and reproduction in response to nutrient availability, but responses of plant growth and reproduction to multiple levels of nutrient enrichment remain unclear. In this study, a factorial field experiment was performed with manipulation of nitrogen (N) and phosphorus (P) availability to examine seed production of the dominant species, Stipa krylovii, in response to N and P addition in a temperate steppe. There were three levels of N and P addition in this experiment, including no N addition (0 g N m⁻² year⁻¹), low N addition (10 g N m⁻² year⁻¹), and high N addition (40 g N m⁻² year⁻¹) for N addition treatment, and no P addition (0 g P m⁻² year⁻¹), low P addition (5 g P m⁻² year⁻¹), and high P addition (10 g P m⁻² year⁻¹) for P addition treatment. Low N addition enhanced seed production by 814%, 1371%, and 1321% under ambient, low, and high P addition levels, respectively. High N addition increased seed production by 2136%, 3560%, and 3550% under ambient, low, and high P addition levels, respectively. However, P addition did not affect seed production in the absence of N addition, but enhanced it under N addition. N addition enhanced seed production mainly by increasing the tiller number and inflorescence abundance per plant, whereas P addition stimulated it by decreasing the plant density yet stimulating height of plants and their seed number per inflorescence. Our results indicate seed production is not limited by P availability but rather by N availability in the temperate steppe, whereas seed production will be increased by P addition when N availability is improved. These findings enable a better understanding of plant reproduction dynamics in the temperate steppe under intensified nutrient enrichment and can inform their improved management in the future.     

Assignment of 1H, 13C and 15N resonances of the reduced human IgG1 CH3 domain

Here we report the NMR resonance assignments for the reduced form of human IgG1 C_H3 domain, a 26 kDa dimer in solution (residues 341–447). The assignments have been deposited in the BioMagResBank with a BMRB accession number of 15204.     

Odor Memory Stability after Reinnervation of the Olfactory Bulb

The olfactory system, particularly the olfactory epithelium, presents a unique opportunity to study the regenerative capabilities of the brain, because of its ability to recover after damage. In this study, we ablated olfactory sensory neurons with methimazole and followed the anatomical and functional recovery of circuits expressing genetic markers for I7 and M72 receptors (M72-IRES-tau-LacZ and I7-IRES-tau-GFP). Our results show that 45 days after methimazole-induced lesion, axonal projections to the bulb of M72 and I7 populations are largely reestablished. Furthermore, regenerated glomeruli are re-formed within the same areas as those of control, unexposed mice. This anatomical regeneration correlates with functional recovery of a previously learned odorant-discrimination task, dependent on the cognate ligands for M72 and I7. Following regeneration, mice also recover innate responsiveness to TMT and urine. Our findings show that regeneration of neuronal circuits in the olfactory system can be achieved with remarkable precision and underscore the importance of glomerular organization to evoke memory traces stored in the brain.