A quantitative real-time PCR method for tissue factor mRNA

BACKGROUND: Tissue factor (TF) is primarily known for its function to initiate blood coagulation. The range of in vivo expression of TF is wide and requires a dynamic assay for monitoring. A general method for TF mRNA quantitation that is dynamic, sensitive and applicable to a variety of experimental systems or clinical situations is therefore desirable. OBJECTIVES: To develop a method for sensitive and dynamic quantitation of TF mRNA in human blood cells. METHODS: TF mRNA expression was analysed and evaluated in monocyte isolations, in whole blood (healthy volunteers and patients scheduled for percutaneous coronary intervention, PCI) and in a panel of human cell lines. RNA was extracted, reverse transcribed and subjected to real-time PCR amplification, according to the TaqMan technology. A TF plasmid was constructed as calibrator of the assay. Two housekeeping genes used as endogenous controls for cDNA quality and integrity were evaluated. RESULTS: The assay was linear by seven orders of magnitude and detected down to 10(2) copies of the TF plasmid. The coefficient of variation was 4% intra-assay and 28% between the assays when using beta2MG as endogenous control. The beta-actin gene expression was induced by treatment with lipopolysaccharide (LPS) in blood leukocytes and could not be used as an endogenous control. However, beta2MG showed only minor variations upon treatment with LPS. The TF mRNA and antigen expression, measured in a Western blot, correlated well (R(2)=0.903) in a panel of 11 human cell lines. CONCLUSIONS: We have established a method for sensitive and dynamic quantitation of TF mRNA in experimental systems and for clinical situations.     

The assessment of early and late radiation injury to the mouse lung using X-ray computerised tomography

X-ray computerised tomography (CT) was performed on the lungs of CBA and C57Bl mice at varying time intervals after 13 and 16 Gy irradiation to the whole thorax. With careful consideration of artefacts associated with lung cross-sectional area and breathing rate, the mean density of the lung was evaluated in Hounsfield units (CT number). In CBA mice, this parameter showed a biphasic increase in lung density with time from irradiation, corresponding to an early phase of radiation pneumonitis and a late phase dominated by pleural effusions. Reduced lung volumes were also seen during the late response and lung compression due to accumulations of pleural fluid is considered a major factor in these observations. C57Bl mice did not develop radiation pneumonitis but appeared to be equally responsive to later radiation-induced increases in lung density. The results obtained from CT-derived densitometry compared well with measurements gained from functional and survival endpoints. X-ray CT provides a sensitive and informative technique for assessing the extent of radiation injury to the mouse lung and is potentially useful for quantifying the counterpart in patients.     

Elevation of guinea pig brain preprodynorphin mRNA expression and hypothalamic-pituitary-adrenal axis activity by “binge” pattern cocaine administration

The endogenous opioid system and the hypothalamic-pituitary-adrenal (HPA) axis have been implicated in many of the neurobiological effects of cocaine. Previous studies in our laboratory showed that “binge” pattern cocaine administration increases preprodynorphin (ppDyn) mRNA levels in the caudate putamen and circulating levels of corticosterone in the rat. The present study extended these findings to guinea pigs, a species known to have a κ opioid receptor profile similar to that of humans. Male guinea pigs were treated with: (a) “binge” pattern cocaine for 7 days (subchronic) (3 × 15 mg/kg/day, hourly, intraperitoneal); (b) “binge” pattern saline for 5 days followed by “binge” pattern cocaine for 2 days (subacute); or (c) “binge” pattern saline for 7 days. Thirty minutes after the final injection, levels of ppDyn mRNA were quantitated in the nucleus accumbens, caudate putamen, frontal cortex, amygdala, hippocampus, and hypothalamus using a solution hybridization RNase protection assay. Regional distribution of ppDyn mRNA levels in the guinea pig brain was similar to that found in rat, with highest levels in the nucleus accumbens and caudate putamen. In the caudate putamen, ppDyn mRNA was significantly increased following either 2 days (38% increase) or 7 days (32% increase) of “binge” pattern cocaine administration as compared to saline-treated controls. No significant changes in ppDyn mRNA levels were found in any other brain region. Both subacute and subchronic “binge” cocaine administration significantly elevated plasma levels of adrenocorticotropin hormone (ACTH) and cortisol. However, the ACTH and cortisol increases were significantly blunted following 7 days of “binge” cocaine administration as compared to 2 days of drug treatment, reflecting the development of HPA tolerance or adaptation to repeated cocaine administration. Thus, the ppDyn mRNA and HPA responses to cocaine in guinea pigs are similar to those observed in rats.     

核磁共振法测定熊去氧胆酸中鹅去氧胆酸的含量

用一般的滴定方法测定熊去氧胆酸中鹅去氧胆酸的含量是很难进行的。NMR方法能有效地进行测定。样品(10～20毫克)溶解在CDCl_3和DMSO-d_6(8:1v/v)的混合溶剂中,马来酸做标准物,以δ=3.8ppm的鹅去氧胆酸C_7质子峰面积计算含量。方法的变异系数是0.45-1.3%,回收率是99.3-100.1%。     

胎盘,羊水早期到成熟阶段的超声图像定量分析研究

自1994年以来,我们应用DFY-Ⅰ型多功能超声定量分析诊断仪,测量组织结构超声图像的分贝(dB),灰阶(GS)值。本文检测分析了157例正常胎盘、羊水测值,结果表明:正常胎盘、羊水早期、中期和晚期的分贝(dB),灰阶(GS)值各阶段存在着明显差异。本资料提示,“定量仪”对超声图像进行定量分析,对正常胎盘、羊水早期到成熟的演变过程提供了诊断依据。     

Evaluation of isotopically labeled internal standards and methods of derivatization for quantitative determination of cocaine and related compounds

Gas chromatography-mass spectrometry (GCMS) is the preferred method for the analysis of drugs/metabolites in biological specimens with use of isotopically labeled analogs of the analytes as internal standards (ISs). An important aspect of the chemical derivatization (CD) for GC-MS analysis is that the CD products derived from the analyte and the selected IS must generate ions suitable for designating the analyte and the IS. These ions should not have significant cross contribution (CC), i.e., IS contribution to the intensities of the ions designated for the analyte, and vice versa. With this in mind, the authors have conducted a search of isotopically labeled analogs of commonly abused cocaine and related compounds (cocaine, norcocaine, benzoylecgonine, cocaethylene, ecgonine, ecgonine methyl ester, anhydroecgonine methyl ester) that are commercially available. These ISs and analytes were derivatized with various groups of reagents, and the CD products were analyzed by GC-MS. MS data are presented in two forms: (1) systematic presentation of fullscan spectra; and (2) tabulation of CC data for ions with potential for designating the ISs and analytes. Many (if not most) of these full-scan spectra are not yet available in the open literature and should be of routine reference value to forensic and clinical laboratories that are engaged in the analysis of these drugs/metabolites. Fullscan MS data were further used to select ion pairs with potential for designating the analytes and ISs in quantitative analysis protocols. The CC data of these ion pairs were evaluated using data collected under the selected ion-monitoring mode and summarized in table format. The data exhibited similar CC characteristics in each alkyl, acetyl, or TMS series. Among the potentially usable ion pairs derived from a specific CD group, there was a trend that the ion pairs with higher mass showed better CC data. The CC data derived from the use of ISs labeled with more deuterium atoms were generally more favorable. These data should save enormous amounts of time and effort for practicing laboratories in their search for optimal analytical parameters.     

Standard methods of electron microscopic radioautography

The standard methods of making electron microscopic radioautograms and quantitation of silver grains in our laboratory were presented. Specialized techniques for insoluble or soluble compounds should be employed for demonstrating the synthesis of various macromolecules and the incorporation of various labeled substances. Specimens for electron microscopic radioautography processed under the same conditions can be employed for the quantitation of radioactive substances in subcellular compartments using image analyzers.     

HBV-DNA定量标准品的构建

目的 建立一种简便快速的DNA定量标准品的制备方法.方法 PCR扩增目的片段,与T载体连接,转化感受态大肠杆菌DH5d,阳性克隆进行测序证实.结果 标准品建立的标准曲线有较大的线性范围,批内和批间重复性也较好,且可长期稳定保存.结论 使用质粒标准品对样本中病毒载量进行测定是一种可行的办法.     

Synapse-to-Neuron Ratio in the Lateral Geniculate Nucleus of Rats Exposed Chronically to Ethanol

Effects of chronic ethanol exposure on synapse-to-neuron ratio in the rat lateral geniculate nucleus were investigated. Male Sprague-Dawley rats were exposed to ethanol, using the Lieber-DeCarli liquid diets, for 4 months starting at the age of 5 weeks. Brains were per-fusion-fixed, and the region containing the dorsal lateral geniculate nucleus was cut into slabs (500 μm thick) that were epoxy resin-embedded. From each rat, three slabs containing the structure were serially sectioned for electron microscopy. Using the double disector method, the study shows an unaltered synapse-to-neuron ratio in ethanol-treated rats when compared with controls. The findings are in agreement with previous studies on the visual system using the seme exposure model. In contrast, a previous study has shown that the synapse-to-neuron ratio in locus ceruleus of ethanol-treated rats is reduced by 50%. Other studies have shown that, whereas the glutamatergic NMDA receptor is very sensitive to ethanol, the kainate/AMPA type of receptor is very much less so. Thus, the difference in ethanol-induced synapse elimination between the two regions may reflect this different sensitivity of the glutamatergic receptors, which are of the kainate/AMPA type in the lateral geniculate nucleus and of the NMDA type in the locus ceruleus.     

人血清载脂蛋白B_(100)火箭免疫电泳测定法

利用顺序超速离心法从正常人血浆分离密度为1.030～1.050g/ml 的低密度脂蛋白免疫家兔获得单价抗载脂蛋白B_(100)血清,在国内建立了人血清载脂蛋白B_(100)火箭免疫电泳测定法。本法灵敏、特异,重复性好。测定载脂蛋白B_(100)的范围为100～880ng。批间重复及批内重复变异系数分别为 2.1～2.4％及1.2～2.7％。回收率为103.4±3.4％。