Versatile frequency domain fitting using time domain models and prior knowledge

An iterative nonlinear least-squares fitting algorithm in the frequency domain using time domain models for quantification of complex frequency domain MR spectra is presented. The algorithm allows incorporation of prior knowledge and has both the advantage of time-domain fitting with respect to handling the problem of missing data points and truncated data sets and of frequency-domain fitting with respect to multiple frequency-selective fitting. The described algorithm can handle, in addition to Lorentzian and Gaussian line-shapes, Voigt and nonanalytic lineshapes. The program allows the user the design of his own fitting strategy to optimize the probability of reaching the global least-squares minimum. The application of the fitting program is illustrated with examples from in vivo ¹H– ³¹P-, and ¹³C-MR spectroscopy.     

血小板源性生长因子对肺纤维母细胞前胶原合成的影响

观察血小板源性生长因子（ＰＤＧＦ）对肺纤维母细胞增殖、胶原合成的影响。以改良Ｋｕｍａｒ法，分离培养了大鼠肺纤维母细胞，并采用ＭＴＴ、免疫细胞化学、原位分子杂交等方法。结果：ＰＤＧＦ对肺纤维母细胞具有促增殖作用，其浓度在１～１０μｇ／Ｌ之间呈剂量依赖性。ＰＤＧＦ作用下的纤维母细胞胞浆内线粒体和粗面内浆网明显增多，树状突起和棘状突起也较对照组增多，ＰＤＧＦ作用２４ｈ，肺纤继母细胞Ⅰ、Ⅲ型前胶原ｍＲＮＡ表达增强，作用４８ｈ胞浆内布满Ⅰ、Ⅲ、Ⅴ型前胶原蛋白，作用７２ｈ，大量前胶原则被分泌到细胞外基质中。结论：ＰＤＧＦ不仅可以促进大鼠肺纤维母细胞增殖、合成代谢旺盛和信息传递活跃，而且使其Ⅰ、Ⅲ、Ⅴ型前胶原合成增加。其调控机制之一是在转录水平上增强了前胶原ｍＲＮＡ表达的结果。     

Quantitation of the inhibitory effect of fibrinogen and its degradation products on fibrin polymerization

Anticlotting activities of fibrinogen and its plasmin degradation products--fragments X,Y and D--have been measured. On the molar basis fragments Y and D are found equally active whereas fragment X acts about 2 times stronger. We suggest that the inhibitory effect depends on a set of specific binding sites characteristic of domain D. As fragment X possesses two such sets its activity is twice as high as that of the single-set fragments Y and D. In spite of its two domains D fibrinogen inhibits clotting much weaker than fragment X. This is probably due to the presence in fibrinogen of large COOH-terminal sections of the two A infinity-chains interfering with the inhibitory effect. The possible role of these A infinity-chain sections in fibrinogen-fibrin system is discussed.     

Rapid induction and quantitation of morphine dependence in the rat by pellet implantation

Four schedules of subcutaneous morphine pellet implantation were developed to render rats rapidly physically dependent on morphine. The schedules included implantation of four morphine pellets over a 3-day period (schedule 1), six morphine pellets over a 3-day period (schedule 2), six pellets over a 7-day period (schedule 3), and ten pellets over a 10-day period (schedule 4). Each morphine pellet contained 75 mg of morphine base. The degree of morphine dependence was quantitated by determining the median effective dose (ED₅₀) of naloxone required to induce the stereotyped jumping response. Hypothermia and weight loss, during abrupt and naloxone-induced withdrawal, were also measured. Rats on schedule 4 exhibited a high degree of dependence on morphine as evidenced by an extremely low naloxone ED₅₀ for the precipitated withdrawal jumping response, whereas schedules 1 and 2 produced a low degree of dependence as shown by high naloxone ED₅₀'s. Further evidence for a high degree of physical dependence on morphine is schedule 4 rats was indicated by their greater loss in body weight and greater hypothermic response after abrupt and after naloxone precipitated withdrawal compared with these responses in the rats in the other three schedules. A correlation was found to exist between naloxone ED₅₀ for the jumping response, body weight loss, and hypothermia observed during naloxone-induced withdrawal in morphine-dependent rats. These studies suggest that the implantation of four morphine pellets in the rat produces a mild degree of dependence and that caution should be exercised when making generalized conclusions about the biochemical correlations involved when four or less number of pellets, each containing 75 mg of morphine base, are used to induce morphine dependence in the rat.     

The 'omics revolution and our understanding of sperm cell biology

The foundations of proteomics are to study gene products and their regulatory roles within cells. Paradoxically, the only evidence that sperm cells make new proteins is through mitochondrial protein synthesis. Yet despite this, spermatozoa are the perfect candidates for mass spectrometry and hence, proteomic analysis. These enterprising cells use a plethora of post-translational modifications in order to gain functionality following their production within the testis. By using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and more recently liquid chromatography-mass spectrometry (LC-MS)/MS, recent advances in sperm cell biology, through the use of proteomics, is making unparalleled progress. The protein inventory lists being generated have shed light on transmembrane proteins, kinases and chaperones never previously recognized. In addition, the ability to isolate either phosphopeptides or glycopeptides and quantify the differences between cells of two different populations make proteomic analysis of spermatozoa a real chance to finally answer some age old questions.     

High-throughput quantitation of nefazodone and its metabolites in human plasma by high flow direct-injection LC-MS/MS

A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine (mCPP) in human plasma. The method utilized d₇-nefazodone, d₇-hydroxynefazodone, d₄-BMS-180492 and d₄-mCPP as internal standards (IS). The plasma samples were injected into the LC–MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis^® HLB column (on-line extraction column, 1 mm × 50 mm, 30 μm) and a conventional Luna C8 column (analytical column, 4.6 mm × 50 mm, 5 μm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC–MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.     

Quantitation of fetal DNA in maternal serum during the first trimester of pregnancy by the use of a DAZ repetitive probe

Cell-free fetal DNA in maternal plasma or serum is at present widely investigated as a source of fetal genetic material, both in studies of pregnancy-related disorders and in planning strategies for non-invasive prenatal diagnosis. Despite the number of trials already performed on the quantitation of fetal DNA, data about the amount of DNA at the beginning of pregnancy, in particular in the first trimester, remain limited. A new probe mapping on the deleted in azoospermia (DAZ) repetitive region of the Yq chromosome was designed for an early assessment of fetal DNA concentration in maternal serum. Among 57 pregnant women prospectively studied in their first trimester, fetal DNA was detected already by the 5th gestational week, with the analysis becoming reliable by the 8th week of gestation when a 100% accuracy in fetal sex determination was achieved. Moreover, in the three cases of pregnancy ending in fetal loss, the amount of fetal DNA apparently decreased before the abortion was diagnosed, whereas it consistently showed an increasing trend in normal pregnancies. Real-time PCR with the use of DAZ multilocus probe can efficiently quantitate free fetal DNA in the maternal serum at the beginning of pregnancy.     

Analysis of soy isoflavone plasma levels using HPLC with coulometric detection in postmenopausal women

A reliable chromatographic method for the determination of soy isoflavones (genistein, daidzein and glycitein) using a coulometric detection has been developed and applied to analyse plasma of postmenopausal women. The chromatographic separation was performed on a C18 reversed phase column with a mobile phase composed of acetonitrile–phosphate buffer mixture. Coulometric detection was carried out at +0.500 V. A careful and rapid solid phase extraction procedure on hydrophilic/lipophilic cartridges was chosen for plasma sample purification with and without hydrolysis obtaining good extraction yield values for all the analytes (>90.0%). The enzymatic hydrolysis step was necessary for the determination of the total amount of soy isoflavones. The limit of quantitation was 0.5 ng mL⁻¹ for genistein and 0.25 ng mL⁻¹ for daidzein and glycitein. The method was found to be precise and accurate. Thus, the proposed method is suitable for the analysis of soy isoflavones (free and total amounts) in plasma of postmenopausal women under treatment with the SoymenGN^® dietary supplement.     

空间环境诱导褪色沙雷菌LCT-SM166的蛋白质组学分析

目的 分析和鉴定神舟八号飞船搭载褪色沙雷菌的差异表达蛋白质。方法 对神州八号飞船搭载的褪色沙雷LCTSM166及地面对照组LCT-SM213 进行分离并进行16sRNA 鉴定,采用同重同位素相对与绝对定量（isobaric tags for relative and absolute quantitation,iTRAQ） 方法对褪色沙雷菌LCT-SM166 和LCT-SM213 进行蛋白质组质谱检测,分析两株菌的差异表达蛋白。结果 共鉴定到1 713 个蛋白质,LCT-SM166 与LCT-SM213 的差异蛋白组学分析发现了111 个蛋白质表达的改变,其中29 个蛋白表达上调,91 个蛋白表达下调。基因本体论（gene ontology,GO） 功能富集分析显示大多数差异蛋白质主要与能量代谢有关。结论 空间环境可影响褪色沙雷菌大量蛋白质的表达,差异表达蛋白主要分布在代谢相关过程。空间诱变菌株的蛋白质组学研究为后续贯穿组学的研究奠定基础。