Rat liver homogenate (S9)-mediated binding of benzo[a]pyren to DNA in V79 cells,V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[a]pyrene (B[a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[a]P (anti-B[a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[a]PDE-DNA adduct to the 9-OH-B[a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[a]P in the presence of the S9 activation system.These results demonstrate that the relative abilities of various reactive bbenzo[a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.     

Superior resolution of gamma-crystallins from microdissected eye lens by cation-exchange high-performance liquid chromatography

A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.     

BIODEGRADATION OF PAHS IN LONG-TERM CONTAMINATED SOIL CULTIVATED WITH EUROPEAN WHITE BIRCH (BETULA PENDULA) AND RED MULBERRY (MORUS RUBRA) TREE

The ability of birch (Betula pendula) and mulberry (Morus rubra), cultivated either separately or together with perennial ryegrass (Lolium perenne), to enhance the biodegradation of 15 selected polycyclic aromatic hydrocarbons (PAHs) in long-term contaminated soil was studied in a greenhouse experiment. The microecosystems (MESs) were cultivated for either 12 or 18 months at a natural photoperiod. The fact that the soil chosen for cultivation had been contaminated for over 50 years was expected to be the main factor limiting biodegradation. Extracts of both planted and unplanted soil were analyzed using HPLC. After 1 year of cultivation, the overall content of the investigated compounds had declined to 50%. The concentrations of fluoranthene and pyrene, the PAHs originally present in the soil in the highest concentrations (103.5 and 83.3 mg/kg, respectively), had decreased to 28.0 and 18.0 mg/kg, respectively. In addition, other compounds were successfully degraded, including even benzo[a]pyrene. Benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were the only PAHs remaining and were almost entirely undegraded. Because few significant differences were found between the various types of MESs studied, degradation can primarily be attributed to the presence of degrading microorganisms in the soil.     

Assay of galactosyltransferase by high-performance liquid chromatography

Galactosyltransferase catalyzes transfer of galactose from UDP-galactose to glucose or N-acetylglucosamine with resultant formation of galactosides and UDP. In this new assay galactosyltransferase activity is measured by determining UDP by isocratic high-performance liquid chromatography on an amino-bonded column monitored spectrophotometrically. Concurrently, unreacted UDP-galactose and breakdown products arising from UDP-galactose (UMP and uridine) are also determined. The new technique does not require radioactive substrates, permits usage of saturating concentrations of UDP-galactose, and provides monitoring of side reactions.     

HPLC测定灵芝子实体水提物及相关产品中三萜的含量

灵芝药品大多以灵芝子实体水提物为原料,为快速准确测定灵芝子实体水提物及相关产品中三萜的含量,建立了具有较好分离效果的HPLC分析测定方法。通过优化色谱柱和洗脱条件,优选出Agilent Zorbax SB-Aq C18色谱柱(250 mm×4.6 mm,5μm),以乙腈-乙酸水溶液(0.01%)为流动相梯度洗脱,流速为1.0 mL/min,检测波长252 nm,柱温30℃,该条件下灵芝酸A、灵芝酸F等10种灵芝酸得到较好的分离。方法学考察显示该分析方法精密度、重复性、稳定性和加样回收率的RSD值均小于5%,可以用于灵芝酸C2、灵芝酸G、灵芝烯酸B、灵芝酸B等10种灵芝酸的定量检测。通过对灵芝子实体原料、水提物和市售灵芝产品中10种三萜类成分分析发现,灵芝子实体水提物中均含有这10种三萜,含量为2.52%–6.83%,较子实体原料大幅提高,市售的灵芝产品中的三萜含量为0.27%–0.84%。该方法的建立为灵芝水提物及其产品质量标准的建立奠定基础。     

Isolation,Purification, and Physical and Chemical Properties of Neuraminidase Inhibitor No. 289

The neuraminidase inhibitor produced by Streptomyces sp. No. 289 has been isolated from a culture filtrate and purified, and the properties of the purified preparation have been investigated. The inhibitor has a molecular weight of about 100,000, being free from neuraminic acid or its analogs and consisting of 88% of sugar and 12% of protein. The sugar constituent is mainly composed of equal amounts of glucose and mannose, and the protein constituent lacks S-containing amino acids. An elementary analysis gives 37.33% C, 6.12% H and 1.29% N. The activity of the inhibitor is stable to heating at 100°C for 10 min and to the actions of various proteolytic enzymes, but is weakened by periodate oxidation. These properties have proved that the inhibitor is completely different from those so far reported.     

Monoamines and their metabolites in somatosensory,visual, and cingulate cortices of adult rat: Differences in content and lack of sidedness

Small areas of somatosensory, visual and cingulate cortex were microdissected and assayed for their monoamine content by high-performance liquid chromatography with electrochemical detection. No differences were found between the right and the left hemisphere for any area nor for any of the monoamines. The values averaged from left and right hemispheres for the sensory areas were significantly different from the cingulate in the content of norepinephrine, 4-hydroxy-3-methoxyphenylglycol, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxyl-tryptophan, serotonin and 5-hydroxyindole-3-acetic acid. The two sensory cortices differed in their levels of norepinephrine, dopamine, 3–4-dihydroxyphenylacetic acid and homovanillic acid. In the latter comparison, the measured amounts were higher in somatosensory than in visual cortex. This biochemical heterogeneity in monoamine distribution may reflect specific innervation patterns for these compounds in these discrete cortical areas and allows differences in content to be related to functional specialization of the cerebral cortex.     

High-throughput,Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.     

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules ($\text{S}{}_{\mathrm{<mtext>H</mtext>}}\text{(}\text{average sedimentation coefficient}\text{) = 12 400}\text{S}$ in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin ($\text{?95\%}$ of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium.The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.     

干扰素-β1b的高效表达、纯化及抗病毒活性研究

IFN-β1b是大肠杆菌产生的17位Cys被Ser替换的人IFN-β的类似物,为了获得高表达,使用了大肠杆菌的偏爱密码子,人工合成了IFN-β-1b基因,插入质粒pBV220中,转化大肠杆菌DH5α.IFN-β1b的制备过程,包括发酵和一系列的纯化步骤.经修饰,IFN-β1b基因在启动子PRPL控制下发酵表达,合成的蛋白质以包涵体的形式存在.培养的细菌经收集、裂解后,将包涵体释放出来,包涵体经含SDS的溶液溶解,DTT还原.纯化过程包括有机溶剂抽提、分子筛层析、脱盐、氧化复性和反相层析,并用旋转蒸发除去有机溶剂.IFN-β-1b在不同种系来源的细胞上显示不同的抗病毒活性.     

Determination of proton dissociation constants by ion-exchange high-performance liquid chromatography

The common methods to determine dissociation constants of solutes, e.g., uv spectrophotometry, potentiometry, and conductimetry, are accurate but require at least 1 nmol of compound. High-performance liquid chromatography (HPLC) allows 1 pmol of a uv-absorbing compound to be detected. By adjusting the polarity of the mobile phase, reverse and normalphase properties of an ion-exchanger can be minimized, resulting in a high correlation between charge and retardation of the solute. Thus, the degree of ionization of several compounds was monitored in mobile-phase compositions of different pH values using cation exchange. The pK values of several pterin derivatives corresponded to those obtained by other methods. In addition, pK values of two unidentified pterin derivatives were determined, using only 20 pmol of each.     

The disposition of venlafaxine enantiomers in dogs, rats, and humans receiving venlafaxine.

A stereospecific high-performance liquid chromatographic (HPLC) method was developed for the quantitation of the enantiomers of venlafaxine, an antidepressant, in dog, rat, and human plasma. The procedure involves derivatization of venlafaxine with the chiral reagent, (+)-S-naproxen chloride, and a postderivatization procedure. The method was linear in the range of 50 to 5,000 ng of each enantiomer per ml of plasma. No interference by endogenous substances or known metabolites of venlafaxine occurred. Studies to characterize the disposition of the enantiomers of venlafaxine were conducted in dog, rat, and human, following oral administration of venlafaxine. The Cmax, area under the curve (AUC) and (S)/(R) concentration ratios of the (R)- and (S)-enantiomers were compared. In rats, the mean plasma ratio of (S)-venlafaxine to that of (R)-venlafaxine over 0.5 to 6.0 h varied from 2.97 to 8.50 with a mean value of 5.51 +/- 2.45. The Cmax, AUC0-infinity, and t 1/2 values of the (R)- and (S)-enantiomers in dogs were not significantly different from one another (P greater than 0.1). The mean ratios [(S)/(R)] of enantiomers of venlafaxine in human over a 2 to 6 h interval ranged from 1.33 to 1.35 with an overall ratio of 1.34 +/- 0.26 (n = 12). These ratios of the enantiomers [(S)/(R)] were not statistically different from unity (P greater than 0.1) indicating that the disposition of venlafaxine enantiomers in humans is not stereoselective and is more similar to that in dogs than that in rats.     

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.     

A new feature of angiotensin-converting enzyme in the brain: hydrolysis of substance P

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.     

Characterization of human alcohol dehydrogenase isoenzymes by high-performance liquid chromatographic peptide mapping

Human liver alcohol dehydrogenase (ADH, EC 1.1.1.1) is a large and heterogeneous family of isoenzymes and the high-performance liquid chromatographic peptide mapping technique which was developed here recognizes differences and similarities between them. Isoenzymes were S-carboxymethylated, digested with trypsin, and the mixtures of tryptic peptides fractionated by reverse-phase gradient chromatography on octadecylsilane columns, using perchlorate-phosphate buffer and acetonitrile as eluants. The resultant peptide maps were reproducible, showing great similarities between the αβγ-ADH isoenzymes (now called Class I) on the one hand and remarkable differences between these and both the π- and χ-ADH isoenzymes (now called Class II and III, respectively) on the other. This implies that these three isoenzyme groups have characteristic primary structures which correspond to their typical substrate specificities and kinetics.     

Biosynthesis of L-[15N]aspartic acid and L-[15N]alanine by immobilized bacteria

Glycoproteins in nitrocellulose transfers of electrophoretically separated mixtures of cellular and viral proteins are rapidly and sensitively located by sequential incubation with the lectin concanavalin A and the enzymatically active glycoprotein horseradish peroxidase. The bound enzyme is located by incubation with a substrate which is converted to a highly insoluble colored product. The specificity of the method is demonstrated by the abolition of concanavalin A binding in the presence of α-methyl mannoside. The method is capable of detecting as little as 60 ng of a purified model glycoprotein after electrophoresis. It has been applied to the analysis of the glycoproteins of purified Lassa virus and of the virus-specific glycoproteins in Japanese encephalitis virus-infected cells.     

Effects of acute handling stress on cerebral monoaminergic neurotransmitters in juvenile Senegalese sole Solea senegalensis

Juvenile Senegalese sole Solea senegalensis were subjected for short periods to two different types of handling‐related stress: air exposure stress and net handling stress. The S. senegalensis were sacrificed 2 and 24 h after the stress events and the levels of serotonin (5‐HT), noradrenaline (NA), dopamine (DA) and their respective major metabolites, 5‐hydroxyindoleacetic acid (5‐HIAA), 3‐methoxy‐4‐hydroxyphenylglycol (MHPG) and 3,4‐dihydroxyphenylacetic acid (DOPAC), were measured in three brain regions (telencephalon, hypothalamus and optic tectum) and compared with those in control, non‐stressed S. senegalensis. Neither type of stress caused any significant alteration of serotoninergic activity (5‐HIAA:5‐HT ratio) or NA levels. Dopaminergic activity (DOPAC:DA ratio) was lower in stressed fish in all of the brain regions studied. For both air exposure stress and net handling stress, DA levels were significantly higher (P < 0·05) than in the control S. senegalensis. In addition, the higher DA levels after net handling stress were always significantly higher (P < 0·05) than those observed after acute air exposure stress, except in the telencephalon after 24 h. The significantly lower DOPAC:DA ratio (P < 0·05) in all of the brain regions studied was only observed in response to net handling stress.     

Carotenoid pigments in male American goldfinches: what is the optimal biochemical strategy for becoming colourful?

Studies of brilliant carotenoid‐based coloration in birds have traditionally centred on the role that these colours play in attracting mates. More recently, biologists have begun to take a biochemical approach to understanding the types of pigments found in feathers and how these relate to the expression of ornamental coloration. Nevertheless, surprisingly few studies have assessed the types and amounts of carotenoids present in the diet or blood of animals in relation to season, sex, condition or sexually attractive colour traits, particularly for wild birds. It is conceivable not only that the total concentration of pigments available is an important predictor of sexual attractiveness and mate quality, but also that specific pigments vary among individuals and play more important physiological and pigmenting roles than others. We investigated the carotenoid content of blood and feathers in wild‐caught, yellow‐pigmented male American goldfinches (Carduelis tristis) throughout the year to determine the optimal biochemical strategy for becoming colourful. We found that birds acquired two main yellow hydroxycarotenoids (lutein and zeaxanthin) from the diet during both moulting and non‐moulting periods. Blood concentrations of both pigments changed significantly over time, with moulting birds accumulating higher levels of both lutein and zeaxanthin, but proportionally more zeaxanthin, than non‐moulting birds. Moulting birds that acquired more lutein and more zeaxanthin in blood deposited a higher concentration of carotenoid pigments (canary xanthophylls A and B) into plumage and acquired more colourful feathers. In sum, these results indicate that (a) the types of dietary carotenoids available across seasons do not change in American goldfinches, (b) seasonal fluctuations in plasma‐carotenoid signatures may result from differences in dietary access or pigment processing, and (c) the best biochemical strategy for becoming a colourful, wild male goldfinch is to accumulate as many dietary/blood pigments as possible during moult. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 273–280.     

人工接种冠突散囊菌对白茶主要呈味物质的影响

本文为了排除其他微生物的干扰,首次以人工接种的方式研究了冠突散囊菌Eurotium cristatum对白茶主要呈味物质的作用。采用高效液相色谱、分光光度计等方法,分析表明冠突散囊菌能够显著降低白茶中呈苦涩味的表没食子儿茶素没食子酸酯（EGCG）和表儿茶素没食子酸酯（ECG）,并提高表没食子儿茶素（EGC）、Asp、His、咖啡碱和山奈酚的含量;灭菌压制的过程中EGCG可能异构化成为更稳定的没食子儿茶素没食子酸酯（GCG）,且二者以相近的含量共存。冠突散囊菌可以降低人工发花白茶饼中呈苦涩味的化合物含量,从而达到减少白茶饼苦涩味的效果;灭菌压制过程也能够降低白茶饼的苦涩味物质的含量。“发花”处理为白茶带来了新的风味,可以丰富白茶产品种类,同时为促进粗老原料白茶的综合利用提供新思路。