HPLCMS/MS法检测中华鳖中磺胺类药物残留

建立了一种液相色谱-电喷雾串联质谱同时测定中华鳖中21种磺胺类药物残留的方法。均质样品先后用乙腈、二氯甲烷提取,合并提取液,取部分提取液经氮吹浓缩。残渣用1mL流动相溶解,饱和正己烷脱脂净化。采用ZORBAX Ec lipse XDB-C8色谱柱,以含0.2%乙酸的水溶液和甲醇（7∶3）为流动相,梯度洗脱,在电喷雾-多反应监测离子模式下,进行定量定性分析。方法的定量限为4μg/kg;以标准加入法计算回收率,在4—40μg/kg添加范围内,平均回收率为75.2%—104%;相对标准偏差为0.16%—9.98%。     

峰胶乙醇提取物化学成分的GC／MS研究

利用气相色谱-质谱联用法(GC-MS),HP-5MS 30 m×0.25 mm×0.25μm5%苯甲基聚硅氧烷弹性石英毛细管柱,对峰胶乙醇提取物化学成分进行了分析,分离出127种组分,采用峰面积归一化定量,鉴定出45种成分,共占其色谱流出组分总量的82.8%,其中萜类、黄酮类化合物约占31%.     

MALDI AND FAB MASS SPECTROMETRY OF NUCLEOSIDE TRIPHOSPHATES: A COMPARATIVE STUDY

Abstract

Comparison of MALDI and FAB mass spectra of dATP, dTTP, dCTP, and dGTP shows that the former technique gives clear molecular ions with minimal fragmentation whereas the latter gives more fragment ions and weaker parent peaks.     

KINETICS STUDY OF THE BIOTRANSFORMATION OF AN OLIGONUCLEOTIDE PRODRUG IN CELLS EXTRACT BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY

The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. The oligonucleotide prodrug was fully metabolized to yield the T₁₂ phosphorothioate likely through a carboxyesterase mediated mechanism.     

Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2: REACTION INTERMEDIATES REVEALED BY CRYSTALLOGRAPHY AND MASS SPECTROMETRY*

Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2.     

Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR*

The susceptibility of the cellular prion protein (PrP^C) to convert to an alternative misfolded conformation (PrP^Sc), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23–231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.     

大鼠杏仁基底外侧核腹侧部向杏仁中央核的纤维投射──HRP逆行示踪结合免疫组织化学法研究

本实验采用ＨＲＰ逆行示踪结合免疫组织化学方法,对大鼠杏仁底基底外侧核腹侧部向中央杏仁核的纤维投射特征及其化学特性进行了研究。一侧杏仁中央核（Ｃｅ）内注射ＨＲＰ后,于双侧杏仁基底外侧核腹侧部（ＢＬＶ）观察到大量ＨＲＰ标记神经元,以对侧为主;在杏仁基底外侧核前（ＢＬＡ）、后（ＢＬＰ）部及梨状皮质内侧部（ＰＣＭ）第Ⅱ、Ⅲ层仅观察到少量ＨＲＰ标记神经元。当注射范围局限于杏仁中央核内侧部（ＣｅＭ）,ＢＬＶ的标记神经元相对多．当注射范围局限于杏仁中央核外侧部（ＣｅＬ）,ＢＬＶ的标记神经元相对少。将有ＨＲＰ标记神经元的切片分别与生长抑素（ＳＯＭ）、脑啡呔（ＥＮＫ）、Ｐ物质（ＳＰ）抗血清按ＡＢＣ法完成免疫组织化学反应,结果在ＢＬＶ、ＰＣＭ第Ⅱ、Ⅲ层观察到ＨＲＰ－ＳＯＭ免疫阳性双标记神经元,但未发现ＨＲＰ－ＳＰ、ＨＲＰ－ＥＮＫ免疫阳性双标记神经元;在ＢＬＡ和ＢＬＰ未发现ＨＲＰ－ＳＯＭ、ＨＲＰ－ＥＮＫ、ＨＲＰ－ＳＰ免疫阳性双标记神经元。本文着重讨论了ＢＬＶ与内脏功能活动的关系,认为ＢＬＶ不同于ＢＬＡ与ＢＬＰ,它参与“内脏环路”。此外,还分析了ＰＣＭ投射到Ｃｅ的神经元的功能学意义。     

LABELLING OF ACETYLCHOLINE IN THE BRAIN OF MICE FED ON A DIET CONTAINING DEUTERIUM LABELLED CHOLINE: STUDIES UTILIZING GAS CHROMATOGRAPHY—MASS SPECTROMETRY

—A method to achieve labelling of the acetylcholine stores of the brain under ideal physiological conditions is described. To this end, mice fed on a choline free diet were supplied with deuterium labelled choline in the drinking water. Labelled and unlabelled choline in plasma and in the brain as well as labelled and unlabelled acetyicholine in the brain were measured by a gas chromatographic-mass spectrometric method. It was found that after 1–25 days on the deuterium choline diet, substantial amounts of the plasma choline and brain acetylcholine were displaced by deuterium choline and deuterium acetylcholine, respectively. Already on the first day, the mole ratio of deuterium choline/total choline in plasma was 0·22, and it approached a maximum of 0·57 on the 14th day. The mole ratios of deuterium acetylcholine/total acetylcholine in the brain were slightly but significantly lower than those of deuterium choline/total choline in plasma 1–14 days, but asymptotically approached the mole ratios of deuterium Ch/total Ch in plasma by 25 days. Intact brains submitted to incubation at room temperature for 10 min increased their total choline content by about 500 per cent. Concurrently, in brains from animals kept on a deuterium choline diet for 1–2 days, the level of deuterium choline rose only by 50 per cent after incubation. Deuterium choline levels increased, however, by 200–300 per cent in the brains from animals kept on the deuterium diet for longer time periods. On the basis of these data it is suggested that: (a) choline in plasma is partly supplied from the food and partly from endogenous sources; (b) plasma choline rapidly equilibrates (less than one day) with a pool of Ch in the brain which is responsible for biosynthesis of acetylcholine; (c) the size of this choline pool is in the order of 34–40 nmol/g.     

An Endogenous Circadian Rhythm in the Rate of Carbon Dioxide Output of Bryophyllum: VI. ACTION SPECTRUM FOR THE INDUCTION OF PHASE SHIFTS BY VISIBLE RADIATION

A comparison has been made of the effectiveness of a 4-h exposureto equal quantum-flux densities of radiation in different zonesof the visible spectrum in shifting the phase of the endogenouscircadian rhythm of carbon dioxide metabolism in leaves of Bryoyihyllumfedtschenkoi. At an incident quantum-flux density of 8.9 ? 10^–13einsteins cm^–2 8^–1, only radiation between 600 and700 nm induced a phase shift, maximum activity being found at(560 nm. At a higher incident quantum-flux density of 1.9 ?10^–11 einsteins cm^–2 8^{– 1}, the peak of activitywas broader and extended from 560 to 700 nm. At both flux densitiesa sharp cut-off occurred at 700 nm. The action spectra are somewhat similar to the absorption spectrumof phytochrome except that they show no minor peak in the bluezone. No evidence has yet been obtained that the inductive effectof red light can be reversed by exposure to far-red radiationas in the case of a typical phytochrome-mediated response. Ultra-violet radiation at 237 nm had no effect upon the phaseof the rhythm.     

A TAXONOMIC STUDY OF THE GENUS XYSTOPHORA (LEPIDOPTERA:GELECHIIDAE) FROM CHINA*

Abstract The present paper deals with species of the genus Xystophora Wocke from China. Four species are described as new to science: Xystophora ingentidentalis sp. nov., Xystophora chengchengensis sp. nov., Xystophora paroisaccula sp. nov. and Xystophora novipsammitella sp. nov. Xystophora carchariella (Zeller) is reported for the first time from China. The genitalia figures of the new species and key to all the known Chinese species are provided.     

Assembly of a Comprehensive Regulatory Network for the Mammalian Circadian Clock: A Bioinformatics Approach

By regulating the timing of cellular processes, the circadian clock provides a way to adapt physiology and behaviour to the geophysical time. In mammals, a light-entrainable master clock located in the suprachiasmatic nucleus (SCN) controls peripheral clocks that are present in virtually every body cell. Defective circadian timing is associated with several pathologies such as cancer and metabolic and sleep disorders. To better understand the circadian regulation of cellular processes, we developed a bioinformatics pipeline encompassing the analysis of high-throughput data sets and the exploitation of published knowledge by text-mining. We identified 118 novel potential clock-regulated genes and integrated them into an existing high-quality circadian network, generating the to-date most comprehensive network of circadian regulated genes (NCRG). To validate particular elements in our network, we assessed publicly available ChIP-seq data for BMAL1, REV-ERBα/β and RORα/γ proteins and found strong evidence for circadian regulation of Elavl1, Nme1, Dhx6, Med1 and Rbbp7 all of which are involved in the regulation of tumourigenesis. Furthermore, we identified Ncl and Ddx6, as targets of RORγ and REV-ERBα, β, respectively. Most interestingly, these genes were also reported to be involved in miRNA regulation; in particular, NCL regulates several miRNAs, all involved in cancer aggressiveness. Thus, NCL represents a novel potential link via which the circadian clock, and specifically RORγ, regulates the expression of miRNAs, with particular consequences in breast cancer progression. Our findings bring us one step forward towards a mechanistic understanding of mammalian circadian regulation, and provide further evidence of the influence of circadian deregulation in cancer.     

A STUDY OF THE BACTERICIDAL ACTION OF ULTRA VIOLET LIGHT : III. THE ABSORPTION OF ULTRA VIOLET LIGHT BY BACTERIA

The simple conclusion of former investigators that the shorter the wave length of ultra violet light the greater the bactericidal action is in error. A study with measured monochromatic energy reveals a characteristic curve of bactericidal effectiveness with a striking maximum between 260 and 270 m.µ. The reciprocal of this abiotic energy curve suggests its close relation to specific light absorption by some single essential substance in the cell. Methods are described for determining the absorption curve, or absorption coefficients, of intact bacteria. These curves for S. aureus and B. coli have important points of similarity and of difference with the reciprocals of the curves of bactericidal incident energy, and point the way in a further search for the specific substance, or substances, involved in the lethal reaction.     

THE MAIN NONPOLAR CHLOROPHYLL c FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) IS A CHLOROPHYLL c 2-MONOGALACTOSYLDIACYLGLYCERIDE ESTER: A MASS SPECTROMETRY STUDY

The main nonpolar chlorophyll c  -like pigment was extracted from Emiliania huxleyi (Lohm.) Hay et Mohler (strain CCMP 370) cultures and isolated by preparative column chromatography and HPLC. The pigment, whose visible spectrum closely resembled that of chlorophyll c  ₂, was studied by low-resolution fast atom bombardment mass spectrometry, showing a very high mass molecular ion (m/z 1313). The fragment ions, either in the direct spectrum or obtained by tandem mass spectrometry with collision-induced dissociation of the molecular ion, were compatible with the consecutive losses of two fatty acids (14:0 and 18:4), glycerol, and a hexose, leaving a chlorophyll c  ₂ backbone, suggesting the molecule consists of a chlorophyll c  ₂ residue linked, via an ester bond, to the sugar moiety of a monohexosyldiacylglycerol. The identities of the two fatty acid residues (14:0 and 18:4n-3) were subsequently corroborated by gas chromatography of the corresponding methyl esters. Chemical hydrolysis–derivatization–gas chromatography–mass spectrometry demonstrated the occurrence of glycerol and that galactose is the constituent sugar. The porphyrin obtained on acid hydrolysis showed chromatographic and visible spectral properties identical to pheoporphyrin c  ₂. This evidence led us to propose a tentative structure whose molecular formula, C₇₆H₉₆O₁₄N₄Mg, was supported by the values of exact mass measurements by high-resolution fast atom bombardment mass spectrometry. This novel structure represents the highest molecular weight natural chlorophyll described to date.     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).     

Discovery of a Novel Function for Human Rad51: MAINTENANCE OF THE MITOCHONDRIAL GENOME*

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.