持家基因作为相对定量内标物的稳定性比较

定量PCR是在PCR定性技术基础上发展起来的核酸定量技术,定量检测时常用编码甘油醛-3-磷酸脱氢酶、β-肌动蛋白、28S和18SrRNA等的持家基因作为内部参照,这些基因被认为在某些类型细胞中的表达是恒定的。近年来,很多研究者发现上述持家基因的表达水平并不稳定,利用它们作为内标来定量并不准确。因此,在进行定量实验时应选择适当的2种或2种以上的内参基因,以减少检测标本间的差异。     

A Rapid Screening Technique for Detection of Diosgenin Through In Situ Cytophotometry

Costus speciosus (Koenig) Sm. contains diosgenin, an important drug in family planning programs in India and underdeveloped countries. A simple, rapid method has been developed for in situ quantitation of diosgenin in this plant. The method is based on the formation of a suitable colored product of diosgenin in frozen sections of fresh material followed by determination of its optical density by in situ cytophotometry. The staining reagent is a combination of anisaldehyde, sulfuric acid and acetic acid. A positive correlation has been observed between cytophotometric diosgenin estimates and those derived from thin layer chromatography of extracts. The method is convenient for routine screening of plants for steroidal sapogenins such as diosgenin.     

Deuterium-labeled Phaseic Acid and Dihydrophaseic Acids for Internal Standards

The concentration of abscisic acid in plants is regulated not only by biosynthesis, but also by metabolism. Abscisic acid is metabolized to phaseic acid via 8′-hydroxyabscisic acid, and phaseic acid is then converted to dihydrophaseic acid and its epimer. A quantitative analysis of these metabolites is important as well as that of abscisic acid to understand changes in the concentration of abscisic acid in plants. However, no internal standards of the metabolites suitable for quantitative analysis have been reported. We prepared 7′-deuterium-labeled phaseic acid with a deuterium content of 86%, using the equilibrium reaction between phaseic acid and 8′-hydroxyabscisic acid. 7′-Deuterium-labeled dihydrophaseic acids were obtained by reducing 7′-deuterium-labeled phaseic acid. The levels of the metabolites in plant organs were determined by using the deuterated metabolites as internal standards.     

A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Cytophotometry of DNA in serial sections]

DNA of leprosy cells in serial paraffine sections stained after Felgen were studied photometrically in scanning microscope SPM with electron computer Wang 720C. Significant differences in extinction values (up to 30--35%) between individual sections within the same series were revealed. Cumulative results of different series were rather uniform. It was concluded that photometric studies of serial sections should be carried out for reliable determination of DNA.     

Internal Standards for Gas Chromatographic Analysis of Metabolic End Products from Anaerobic Bacteria

2-Methylpentanoic acid and benzoic acid are suggested for use as routine internal standards for gas chromatographic analysis of microbial end products.     

A Comparison of Washing Methods for Hair Mineral Analysis: Internal Versus External Effects

A major difficulty in hair elemental (mineral) analysis for biomonitoring is adequate understanding of the effectiveness of washing procedures. A review of washing protocols used in hair analysis publications showed little consensus with regard to solvents and surfactants used, washing times, and number of washing stages. Two washing approaches were subsequently used to compare their influence on internal and external surface elemental signals determined with time-of-flight secondary ion mass spectrometry. Na, K, Ca, Mg, and Fe were assessed with regard to their relative signal compared to carbon. Both washing methods had similar effect. All elements except for Fe appear to be removed from the surface of the hair as well as from inside the hair. Only the internal Fe content changed with washing and could indicate that external surface bound Fe may not be removed with most washing procedures. It is shown that washing procedures can have a significant effect on reducing the internal elemental signal levels in hair.     

^18O内标质谱法比较定量蛋白质组学计量的数学算法

18O内标蛋白质质谱定量分析是一种极具前景的比较蛋白组学研究技术,虽然该方法与迄今所有内标法相比有一系列优点,但是由于其质谱是由16O未标记品种,单及双18O标记品种分子同位素峰簇相互迭加所构成的复杂谱,定量分析的关键是发展有效的质谱解析算法。有鉴于此,综述和讨论了18O内标质谱差异比较蛋白质分析过程中,分子同位素簇分布的简化计算方法,以及质谱混和谱解析的数学方法。     

The Scaling of Plant Height: A Comparison Among Major Plant Clades and Anatomical Grades

The scaling plant height h (m) with respect to stem diameterd (m) was determined for a total 610 species (mosses, n = 40;pteridophytes, n = 16; dicotyledonous herbs, n = 117; palms,n = 17; gymnosperms, n = 105; dicotyledonous trees, n = 315);axial length or mass vs. d was determined for the pteridophytePsilotum nudum ; and the scaling of critical buckling heighth_crit of gymnosperm and dicotyledonous trees was calculatedbased on the record trunk d and average Young's modulus E anddensity p of 33 wood species. The scaling exponents (based onleast squares and reduced major axis regressions;     

Cytophotometry of breast carcinoma. Acridine-orange DNA microfluorimetry with Giemsa counterstain

Investigations have suggested that a correlation exists between DNA ploidy levels and prognosis in human breast carcinoma. Nuclear DNA content can be studied by flow cytometry or cytophotometric analysis. While both methods yield comparable results for DNA distribution, cytophotometry has the advantage of permitting both quantitative cell measurements and cytomorphologic identification of tumor cells. Microfluorimetric analysis of nuclear DNA content was carried out on acridine-orange-stained imprint smears of malignant breast tumors, with the DNA values plotted as a histogram distribution. Quantitative fluorescence measurements of breast carcinoma cells using the acridine-orange stain appeared to be a fairly rapid and simple method for DNA determination as compared to Feulgen DNA analysis. Following cytometric measurements, imprint smears were counterstained by the Giemsa stain and examined by cytomorphologic criteria. With the Giemsa counterstain, the same cytologic preparation could be studied both by quantitative cell measurements and by conventional cytomorphologic criteria. Results are illustrated, and possible implications of the use of this method in the study of tumor behavior and the diagnosis by cytologic methods are discussed.     

Restoring Wetland Plant Diversity: A Comparison of Existing and Adaptive Approaches

Most restoration projects are not designed or assessed in ways that identify cause–effect relationships.When plants die, even detailed postmortem examinations cannot pinpoint causes; e.g., mortalities of 7% vs. 90% in two salt marsh transplantation projects were attributable to differences in hypersalinity and sedimentation, but other effects could not be ruled out. Adaptive restoration (the experimental testing of alternative approaches in restoration sites), however, can clarify cause–effect relationships, while simultaneously restoring plant diversity and informing future restoration efforts. Projects in Tijuana Estuary (California) and Greene Prairie (Wisconsin) demonstrate the approach: (1) A large field experiment at Tijuana Estuary showed that species-rich plantings of halophytes accelerated the development of ecosystem structure and function (over single-species plantings) while simultaneously vegetating an intertidal plain. The six-species assemblages produced more complex canopies and accumulated more biomass and nitrogen than singlespecies and unplanted plots. (2) Also at Tijuana Estuary, an experiment is testing the ability of tidal creek networks to accelerate revegetation and increase food-web support (via increased growth of plants, invertebrates, and fish) in an 8-ha project that simultaneously restored tidal flushing. (3) In Greene Prairie, the ability to establish 33 native species is being tested as replacements for an invasive grass (Phalaris arundinacea). In each case, the adaptive approach informs both the science and practice of restoration. Without experimentation, restorationists are hard-pressed to explain past mortality and to suggest better methods for restoring structure and function. Adaptive restoration can provide the knowledge required, especially when large projects are implemented as sequential modules with experiments that sequentially provide essential information.     

Feulgen Cytophotometry of Pine Nuclei; Effects of Fixation, Role of Formaline

The effects of 5 fixatives: FAA, Carnoy's, Craf III, formalin and glutaraldehyde were analyzed for use in quantitative Feulgen cytophotometry of pine embryo tissues. Craf III and glutaraldehyde had serious deficiencies because they depressed the absorption peak, severely interfered with DNA extraction and in the case of glutaraldehyde there was considerable cytoplasmic dye-binding. Neutral 10% formalin gave good tissue fixation but did not permit the degree of enzymatic or acid extraction of DNA as did Carnoy's solution. Haupt's adhesive, with the usual 4% formalin as a hardener, at temperatures of 45-56 C completely prevented the enzymatic extraction of nuclear DNA by DNase and also greatly increased the resistance of the DNA to mineral acid hydrolysis. Denaturation of DNA by formalin appeared to be responsible for these results. Absorption was linearly related to both section thickness and DNA concentration per nucleus.