滨蒿内酯对哮喘豚鼠气管平滑肌细胞RyR2 mRNA表达影响

研究哮喘豚鼠气管平滑肌细胞Ryanodine受体亚型的变化及滨蒿内酯对其的影响。将动物分为3组:正常组、哮喘组和滨蒿内酯组,采用卵蛋白致敏复制豚鼠哮喘模型,滨蒿内酯组雾化吸入滨蒿内酯。应用RT-PCR鉴定豚鼠气管平滑肌细胞(TSMC)中RyR亚型分布并观察各组豚鼠气管平滑肌细胞RyR2 mRNA表达的变化。豚鼠TSMC表达RyR2和RyR3两个亚型;哮喘时豚鼠TSMC中RyR2 mRNA表达量(以RyR2区带与-βactin区带的密度百分比表示)为47.15%±15.30%,明显低于对照组(77.12%±6.16%)(P<0.01);滨蒿内酯组豚鼠TSMC中RyR2 mRNA量为63.77%±9.09%,较哮喘组增强(P<0.05)。豚鼠TSMC表达RyR2和RyR3两个亚型;哮喘时TSMC RyR2 mRNA表达下调;滨蒿内酯可使哮喘豚鼠TSMC RyR2表达水平得以恢复。     

莴笋地蜘蛛优势种的空间生态位研究

通过对莴笋地蜘蛛优势种的空间生态位研究,发现蜘蛛优势种在每天的上午、中午和下午的空间生态宽度指数值和生态位重叠指数值大小均有较大变化,下午的蜘蛛生态位宽度指数值和生态位重叠指数值较大,上午的次之,中午的最小。     

检测EB病毒TK激酶抗体方法的建立及其在鼻咽癌诊断中的应用

目的:建立检测鼻咽癌(NPC)患者血清中EB病毒TK激酶的酶免疫测定(EIA)方法,用于筛选和辅助诊断早期NPC患者。方法:用基因工程高效表达的EB病毒TK激酶作为包被抗原,建立EIA检测方法,检测NPC患者和对照血清中的TK/IgG抗体。结果:在NPC患者血清中检出了特异的对EB病毒TK激酶的IgG抗体,而正常人血清中为抗体阴性。结论:首先建立了简便、快速、特异和敏感的早期诊断NPC的EIA方法,并作为我公司产品扩展应用。     

Near-infrared fluorescent deoxyglucose analogue for tumor optical imaging in cell culture and living mice

2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has extensively been used for clinical diagnosis, staging, and therapy monitoring of cancer and other diseases. Nonradioactive glucose analogues enabling the screening of the glucose metabolic rate of tumors are of particular interest for anticancer drug development. A nonradioactive fluorescent deoxyglucose analogue may have many applications for both imaging of tumors and monitoring therapeutic efficacy of drugs in living animals and may eventually translate to clinical applications. We found that a fluorescent 2-deoxyglucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), can be delivered in several tumor cells via the glucose transporters (GLUTs). We therefore conjugated D-glucosamine with a near-infrared (NIR) fluorphor Cy5.5 and tested the feasibility of the Cy5.5-D-glucosamine (Cy5.5-2DG) conjugate for NIR fluorescence imaging of tumors in a preclinical xenograft animal model. Cy5.5-2DG was prepared by conjugating Cy5.5 monofunctional N-hydroxysuccinimide ester (Cy5.5-NHS) and D-glucosamine followed by high-performance liquid chromatography purification. The accumulation of Cy5.5-2DG and Cy5.5-NHS in different tumor cell lines at 37 and 4 degrees C were imaged using a fluorescence microscope. Tumor targeting and retention of Cy5.5-2DG and Cy5.5-NHS in a subcutaneous U87MG glioma and A375M melanoma tumor model were evaluated and quantified by a Xenogen IVIS 200 optical cooled charged-coupled device system. Fluorescence microscopy imaging shows that Cy5.5-2DG and Cy5.5-NHS are taken up and trapped by a variety of tumor cell lines at 37 degrees C incubation, while they exhibit marginal uptake at 4 degrees C. The tumor cell uptake of Cy5.5-2DG cannot be blocked by the 50 mM D-glucose, suggesting that Cy5.5-2DG may not be delivered in tumor cells by GLUTs. U87MG and A375M tumor localization was clearly visualized in living mice with both NIR fluorescent probes. Tumor/muscle contrast was clearly visible as early as 30 min postinjection (pi), and the highest U87MG tumor/muscle ratios of 2.81 +/- 0.10 and 3.34 +/- 0.23 were achieved 24 h pi for Cy5.5-2DG and Cy5.5-NHS, respectively. While as a comparison, the micropositron emission tomography imaging study shows that [18F]FDG preferentially localizes to the U87MG tumor, with resulting tumor/muscle ratios ranging from 3.89 to 4.08 after 30 min to 2 h postadministration of the probe. In conclusion, the NIR fluorescent glucose analogues, Cy5.5-2DG and Cy5.5-NHS, both demonstrate tumor-targeting abilities in cell culture and living mice. More studies are warranted to further explore their application for optical tumor imaging. To develop NIR glucose analogues with the ability to target GLUTs/hexokinase, it is highly important to select NIR dyes with a reasonable molecular size.     

Maize uroporphyrinogen III methyltransferase: overexpression of the functional gene fragments in Escherichia coli and one-step purification

S-Adenosyl-L-methionine: uroporphyrinogen III methyltransferase (SUMT), a key regulatory enzyme, converts uroporphyrinogen III to precorrin-2 in the porphinoids biosynthesis. In this study, the mature SUMT was signified that the maize SUMT precursor encoded by the open reading frame of maize SUMT cDNA was deleted the first 91 amino acids constituting the postulated signal peptide. Several mature SUMT fusion and deletion mutants were conducted. It actively expressed in Escherichia coli that the mature SUMT, or the truncated one deleting the C-terminal extra 52 amino acids based on SUMT sequence comparisons. On the contrary, it expressed as an inclusion body in E. coli that the mature SUMT fusion mutant, the SUMT precursor, or the mature SUMT deleting the N-terminal 36 amino acids including glycine-rich region involved directly in SAM binding. The purified His6-tagged mature SUMT was homodimer with a molecular weight of 34 kDa, as shown by SDS-PAGE, 52 kDa using gel-filtration chromatography, and 79 kDa by dynamic light scattering assay. Red fluorescent compounds were associated with the recombinant mature SUMT which were identified as sirohydrochlorin and trimethylpyrrocorphin by spectroscopic analysis. This association slightly altered the protein secondary structure confirmed by circular dichroism assay.     

99mTc-labeled cyclic RGDfK dimer: initial evaluation for SPECT imaging of glioma integrin alphavbeta3 expression

This report describes the evaluation of biodistribution properties of three radiotracers, [(99m)Tc(SQ168)(EDDA)], [(99m)Tc(SQ168)(tricine)(PDA)], and [(99m)Tc(SQ168)(tricine)(TPPTS)] (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-d-Phe})-cyclo{Lys-Arg-Gly-Asp-d-Phe}; EDDA = ethylenediamine-N,N'-diacetic acid; PDA = 2,5-pyridinedicarboxylic acid; TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate), and their potential to image the glioma integrin alpha(v)beta(3) expression in BALB/c nude mice bearing the U87MG human glioma xenografts. It was found that all three radiotracers were able to localize in glioma tumors with a relatively high tumor uptake and long tumor retention time by binding to the integrin alpha(v)beta(3) expressed on both tumor cells and endothelial cells of tumor neovasculature. It seems that the coligand has minimal effect on integrin alpha(v)beta(3) targeting capability of the (99m)Tc-labeled RGDfK dimer, but it has a significant impact on their biodistribution properties. For example, the complex [(99m)Tc(SQ168)(tricine)(TPPTS)] has the lowest liver uptake and the highest metabolic stability in normal BALB/c nude mice. Results from SPECT imaging studies show that the glioma tumors can be clearly visualized with all three radiotracers at 4 h postinjection. Among the three radiotracers evaluated in this study, [(99m)Tc(SQ168)(tricine)(TPPTS)] has the best imaging quality and is a promising candidate for more preclinical evaluations in the future.     

Comparative genomics of emerging human ehrlichiosis agents

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens.     

Sensitive HPLC-APCI-MS method for the determination of cyclovirobuxine D in human plasma

A sensitive high performance liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry (HPLC-APCI-MS) assay for determination of cyclovirobuxine D (CVB-D) in human plasma using mirtazapine as internal standard (I.S.) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C(18) column with a mobile phase of 30 mM ammonium acetate buffer solution containing 1% formic acid-methanol (48:52, v/v). CVB-D was determined with atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS). HPLC-APCI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at [M+H](+)m/z 403.4 for CVB-D and [M+H](+)m/z 266.2 for I.S. Calibration curves were linear over the range 10.11-4044 pg/ml. The lower limit of quantification was 10.11 pg/ml. The intra- and inter-run variability values were less than 9.5 and 12.4%, respectively. The mean plasma extraction recovery of CVB-D was in the range of 85.3-92.8%. The method was successfully applied to determine the plasma concentrations of CVB-D in Chinese volunteers.     

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.     

Immunological thresholds in neurological gene therapy: highly efficient elimination of transduced cells might be related to the specific formation of immunological synapses between T cells and virus-infected brain cells

First-generation adenovirus can be engineered with powerful promoters to drive expression of therapeutic transgenes. Numerous clinical trials for glioblastoma multiforme using first generation adenoviral vectors have either been performed or are ongoing, including an ongoing, Phase III, multicenter trial in Europe and Israel (Ark Therapeutics, Inc.). Although in the absence of anti-adenovirus immune responses expression in the brain lasts 6-18 months, systemic infection with adenovirus induces immune responses that inhibit dramatically therapeutic transgene expression from first generation adenoviral vectors, thus, potentially compromising therapeutic efficacy. Here, we show evidence of an immunization threshold for the dose that generates an immune response strong enough to eliminate transgene expression from the CNS. For the systemic immunization to eliminate transgene expression from the brain, > or = 1 x 10(7) infectious units (iu) of adenovirus need to be used as immunogen. Furthermore, this immune response eliminates >90% of transgene expression from 1 x 10(7)-1 x 10(3) iu of vector injected into the striatum 60 days earlier. Importantly, elimination of transgene expression is independent of the nature of the promoter that drives transgene expression and is accompanied by brain infiltration of CD8(+) T cells and macrophages. In conclusion, once the threshold for systemic immunization (i.e. 1 x 10(7) iu) is crossed, the immune response eliminates transgene expression by >90% even from brains that receive as little as 1000 iu of adenoviral vectors, independently of the type of promoter that drives expression.